USHER syndrome-related gene modifications

Abstract

This disclosure relates to compositions, systems, and methods associated with repair RNA sequences, and related systems for trans-splicing target nucleic acids using repair RNA sequences. Various compositions and systems are described herein, including compositions comprising a repair RNA sequence containing one or more exons and / or introns, a splicing donor, and / or a splicing acceptor, wherein the repRNA is adapted for trans-splicing. The compositions, systems, and methods described herein are suitable for targeting one or more Usher syndrome-related genes and for treating Usher syndrome.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 519,830, filed August 15, 2023; U.S. Provisional Application No. 63 / 602,531, filed November 24, 2023; and U.S. Provisional Application No. 63 / 554,908, filed February 16, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to compositions, systems, and methods for trans-splicing target nucleic acids containing repair RNA.

[0004] Description of text files submitted electronically

[0005] This application contains a sequence list, which is submitted via EFS-Web in XML format. An XML copy named “AMR-012PC_134241-5012_Sequence Listing” was created on August 15, 2024, and is 1,961,984 bytes in size; its contents are incorporated herein by reference in their entirety. Background Technology

[0006] Usher syndrome is a disorder typically characterized by partial or complete hearing and vision loss that worsens over time. Usher syndrome encompasses three sensory dysfunctions: retinitis pigmentosa, congenital hearing loss, and unstable vestibular function. It is classified into one of three categories (Type I, Type II, or Type III) based on the severity of the patient's phenotype. These types are distinguished according to the severity of hearing loss, the presence or absence of balance problems, and the age at which signs and symptoms appear. These types are further subdivided based on their genetic origin. Type I Usher syndrome is generally considered the most severe form: it usually presents with severe hearing loss at birth and vision loss in early development. Type II Usher syndrome (USH2) typically affects balance, usually presenting as milder early-onset hearing loss and vision loss that worsens during puberty. Type II Usher cases account for approximately 50% of all Usher cases. The rarest form is Type III, which can begin around puberty with loss of night vision and hearing loss in late childhood.

[0007] To date, clinicians have explored various approaches to treat Usher syndrome, such as using dual-vector systems or suboptimal CRISPR tools that can be loaded into a single AAV vector to target Usher syndrome. Tools outside the CRISPR domain, such as stem cell therapies and antisense oligonucleotides, have also been investigated. However, all these approaches remain problematic due to undesirable off-target effects, inefficiencies, or combinations with limited applicability.

[0008] Therefore, there is a need for new and reliable gene therapies without off-target effects, as well as related methods, such as for the treatment of Usher syndrome. Summary of the Invention

[0009] Therefore, this disclosure provides, in several aspects, a composition comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing.

[0010] In addition, this disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA, wherein the repRNA comprises: (a) one or more exons and / or introns; and (b) a splicing donor and / or splicing acceptor.

[0011] In one embodiment, the one or more exons are or comprise one or more exons of the target nucleic acid molecule. In another embodiment, the one or more introns are or comprise one or more introns of the target nucleic acid molecule. In yet another embodiment, the one or more exons are or comprise one or more exons of the target nucleic acid molecule. In yet another embodiment, the one or more introns are or comprise one or more introns of the target nucleic acid molecule.

[0012] In one embodiment, the repRNA comprises one or more binding motifs that guide and / or hybridize the repRNA to a target nucleic acid molecule. In another embodiment, the one or more binding motifs bind and / or hybridize to the target nucleic acid molecule indirectly or directly. In yet another embodiment, the one or more binding motifs comprise an antisense sequence to the target nucleic acid molecule. In yet another embodiment, the one or more binding motifs hybridize to exons and / or introns or fragments thereof of the target nucleic acid. In yet another embodiment, the one or more binding motifs hybridize to fragments of exons of the target nucleic acid. In yet another embodiment, the one or more binding motifs hybridize to fragments of introns of the target nucleic acid.

[0013] In the embodiments, the one or more binding motifs comprise about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0014] In the embodiments, the one or more binding motifs are approximately 10-500 nucleotides, 15-500 nucleotides, 20-500 nucleotides, 30-500 nucleotides, 40-500 nucleotides, 50-500 nucleotides, or 60-500 nucleotides, or 70-500 nucleotides, or 80-500 nucleotides, or 90-500 nucleotides, or 100-500 nucleotides, or 100-400 nucleotides, or 100-300 nucleotides, or 100-200 nucleotides, or 200-400 nucleotides, or 200-300 nucleotides, or 300-400 nucleotides, or up to The number of nucleotides is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 150, 200, 250, 300, 400, or 500.

[0015] In embodiments, the compositions or systems disclosed herein comprise one or more sequences that bind to and / or hybridize with RNA-binding peptides. In embodiments, the sequences binding to the RNA-binding peptides are assembled into a secondary structure suitable for interacting with the RNA-binding peptides. In embodiments, the secondary structure is or comprises a hairpin. In embodiments, the secondary structure is or comprises a stem, inner loop, multi-branched loop, or pseudoknot.

[0016] In the implementation scheme, the RNA-binding protein is a viral protein.

[0017] In an embodiment, the RNA-binding polypeptide is any RNA-binding polypeptide, optionally selected from MS2 capsid protein (MCP), PP7 capsid protein, PRR1, HgaII, Qβ capsid protein, IN protein, SLBP (stem-loop histone mRNA-binding protein), and M protein or variants thereof. In an embodiment, the RNA-binding protein is MS2. In an embodiment, the RNA-binding protein is PP7 capsid protein. In an embodiment, the RNA-binding protein is PRR1. In an embodiment, the RNA-binding protein is HgaII. In an embodiment, the RNA-binding protein is Qβ capsid protein. In an embodiment, the RNA-binding protein is IN protein or SLBP protein. In an embodiment, the RNA-binding protein is M protein.

[0018] In an embodiment, the one or more binding motifs include a recognition sequence for the formation of the ribonucleoprotein (RNP) complex.

[0019] In embodiments, the composition or system further comprises a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to snRNA or snoRNA), or a nucleic acid encoding the protein forming or within the RNP. In embodiments, the one or more binding motifs (e.g., but not limited to snRNA or snoRNA), the protein forming or within the RNP, the protein within the RNP, and / or the nucleic acid encoding the protein forming or within the RNP contains modifications or mutations that attenuate, weaken, reduce, decrease, or eliminate RNP activity compared to an unmodified form, and / or result in attenuated RNA modification activity, the RNP activity optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation. In an embodiment, the one or more binding motifs (e.g., but not limited to snRNA or snoRNA), the protein forming the RNP or within the RNP, and / or the nucleic acid encoding the protein forming the RNP or within the RNP contains modifications or mutations that increase, stimulate, or enhance RNP activity or enhance RNA modification activity compared to the unmodified form, wherein the RNP activity is optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation.

[0020] In embodiments, the composition or system comprises repair RNA (repRNA) and / or a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to snRNA or snoRNA), or nucleic acid encoding the protein forming or within the RNP, and / or a small RNA inducing cleavage of RNA and / or CRISPR-Cas enzymes. In embodiments, when the method of the invention is performed in cis or trans as described herein, the composition or system comprises repair RNA (repRNA) and / or a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to snRNA or snoRNA), or nucleic acid encoding the protein forming or within the RNP, and / or a small RNA inducing cleavage of RNA and / or CRISPR-Cas enzymes. In embodiments, cleavage is initiated by an RNP formed on the repRNA, or by an RNP formed in cis or trans.

[0021] In the implementation, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding peptides, and is described herein as "grepRNA".

[0022] In one embodiment, the composition or system includes a splice acceptor. In another embodiment, the composition or system includes a splice donor.

[0023] In one embodiment, (a) at least one intron sequence, (b) a splice acceptor and / or splice donor sequence, and (c) at least one exon sequence are provided in cis or trans, or are suitably provided in cis or trans. In another embodiment, (a) at least one intron sequence, (b) a splice acceptor and / or splice donor sequence, and (c) at least one exon sequence are provided in trans, or are suitably provided in trans. In another embodiment, these elements are controlled by one or more promoters. In another embodiment, these elements are controlled by different promoters. In another embodiment, these elements are operatively linked but separated by cleavable sequences (e.g., self-cleaving ribozymes). In another embodiment, (i) multiple repRNA populations are controlled by different promoters, or (ii) a repRNA and another system member are controlled by different promoters.

[0024] In an embodiment, the one or more binding motifs comprise sequences from small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises sequences from the snRNA or the snoRNA. In an embodiment, the snRNA comprises U1, U2, U3, U4, U5, U6, U7, U8, U9, U10, or U11, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitution, addition, or deletion. In the embodiments, the snRNA or snoRNA is selected from any of SEQ ID NO: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion, optionally wherein any of SEQ ID NO: 144-802, or a fragment or variant thereof, forms an RNP complex, said fragment or variant optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0025] In the implementation scheme, the snRNA or snoRNA targets one or more exon splice enhancers (ESEs), one or more intron splice enhancers (ISEs), one or more exon splice silencers (ESSs), and / or one or more intron splice silencers (ISSs). In the implementation scheme, the snRNA or snoRNA is modified to include at least one or more exon splice enhancers (ESEs), at least one or more intron splice enhancers (ISEs), at least one or more exon splice silencers (ESSs), and / or at least one or more intron splice silencers (ISSs).

[0026] In embodiments, the composition or system comprises repair RNA (repRNA) and a small RNA that induces RNA cleavage. In embodiments, the small RNA that induces RNA cleavage is one or more of the following: siRNA, small hairpin RNA (shRNA), U7 snRNA, U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and antisense oligonucleotides (ASO). In embodiments, the composition or system comprises repair RNA (repRNA), and the small RNA contains a modification or mutation that attenuates, weakens, reduces, diminishes, or eliminates activity compared to the unmodified form. In embodiments, the composition or system comprises repair RNA (repRNA), and the small RNA contains a modification or mutation that increases, stimulates, or enhances activity compared to the unmodified form. In embodiments, when the method of the invention is performed in cis or trans as described herein, the composition or system comprises repair RNA (repRNA) and a small RNA that induces RNA cleavage.

[0027] In the implementation scheme, the snRNA or snoRNA contains N 6-Methyladenosine (M6A) modification. In embodiments, when the method of the invention is performed in cis or trans configuration as described herein, the snRNA contains M6A modification. In embodiments, the snRNA or snoRNA is modified to contain at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence. In embodiments, the snRNA or snoRNA is modified not to contain M6A sites. In embodiments, the repRNA contains at least one or more M6A sites. In one embodiment, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the exon sequence. In another embodiment, the repRNA does not contain M6A sites.

[0028] In one embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence increases the trans-splicing efficiency of the target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In yet another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence decreases the trans-splicing efficiency of the target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In yet another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence increases the trans-splicing efficiency of off-target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In the implementation, compared to the unmodified form, the at least one intron spacer sequence containing at least one ISE and ESS sequence reduces the efficiency of off-target RNA trans-splicing.

[0029] In one embodiment, the repRNA comprises ESS, ESE, ISS, and / or ISE sequences. In another embodiment, the repRNA targets one or more of ESS, ESE, ISS, and / or ISE. In another embodiment, interactions, regulation, and / or binding with one or more of ESS, ESE, ISS, and / or ISE reduce or eliminate the interaction, regulation, and / or binding of one or more of ESS, ESE, ISS, and / or ISE to the target. In another embodiment, the repRNA comprises exon sequences having ESE and ESS sequences. In another embodiment, the exon sequences having ESE and ESS sequences increase or decrease trans-splicing efficiency against the RNA target compared to the unmodified form. In another embodiment, the repRNA comprises exon sequences having ESE and ESS sequences. In another embodiment, the repRNA comprising exon sequences having ESE and ESS sequences increases or decreases trans-splicing efficiency against RNA off-target effects compared to the unmodified form. In another embodiment, the repRNA comprises at least one or more G4 structures. In one embodiment, the repRNA comprises at least one or more G4 structures that isolate SD / SA motifs. In another embodiment, the G4 structure is unwound, for example by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., an endogenous target or exogenously delivered trigger RNA). In this embodiment, the G4 structure reduces off-target effects compared to the unmodified form.

[0030] In one embodiment, the repRNA includes a modification comprising at least one or more scaffold sequences. In another embodiment, the at least one or more scaffold sequences mediate (e.g., recruit) condensate-like aggregation and / or improve the local concentration of the repRNA compared to the unmodified form and other target proteins and / or RNAs. In yet another embodiment, the repRNA includes a modification comprising at least one or more sequences to target the repRNA to the promoter of a target gene of interest, or to a proximal condensate that may contain the promoter. In yet another embodiment, the one or more sequences comprise enhancer RNA, snRNA, and / or snoRNA sequences.

[0031] In one embodiment, the repRNA contains a modification. In another embodiment, the modification improves the interaction and localization with the non-template strand DNA sequence of the target gene compared to the unmodified form. In another embodiment, the non-template strand DNA sequence of the target gene is a promoter, intron, exon, or enhancer. In yet another embodiment, the modification improves the interaction and localization with the non-template strand DNA sequence of the target gene via a protein-directed (e.g., transcription factor, dCas, ZNF, or other RBP) or nucleotide-directed (e.g., R-loop) approach compared to the unmodified form.

[0032] In one embodiment, the repRNA includes a modification comprising an additional RNA element. In another embodiment, the additional RNA element improves subnuclear localization to nuclear spots compared to the unmodified form, thereby enhancing trans-splicing efficiency. In another embodiment, the additional RNA element comprises NEAT1 and / or MALAT1 or fragments thereof. In another embodiment, the additional RNA element comprises the nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitution, addition, or deletion. In another embodiment, the repRNA includes a modification enabling it to target the transcription site of the target RNA. In another embodiment, the repRNA includes a modification comprising a 5' UTR or a 3' UTR modification. In the implementation, the modification containing 5' UTR or 3' UTR alters intracellular or nuclear localization based on interaction with endogenously or exogenously supplied molecules (e.g., the interaction of RNA G4 with transcription factors or other proteins localized to specific cellular compartments).

[0033] In one embodiment, the repRNA contains a modification in its 5' UTR. In another embodiment, the modification in the 5' UTR of the repRNA increases stability compared to the unmodified form. In yet another embodiment, the modification in the 5' UTR of the repRNA decreases stability compared to the unmodified form. In yet another embodiment, the modification in the 5' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form. In another embodiment, the repRNA contains a modification in its 3' UTR. In yet another embodiment, the modification in the 3' UTR of the repRNA increases stability compared to the unmodified form. In yet another embodiment, the modification in the 3' UTR of the repRNA decreases stability compared to the unmodified form. In yet another embodiment, the modification in the 3' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form.

[0034] In an implementation scheme, the repRNA includes a modification comprising modifying the repRNA to include a G4 structure, the G4 structure mediating the recruitment of splice-related RBPs.

[0035] In an embodiment, the repRNA includes modifications that include at least one or more foothold switches in the repRNA. In an embodiment, the at least one or more foothold switches in the repRNA are conditionally activated or deactivated (e.g., SD / SA blocking, binding motif blocking, or RBP blocking) upon detection of endogenously or exogenously supplied target RNA.

[0036] In one embodiment, the repRNA includes a modification comprising at least one or more complementary riboregulators (cis) in the repRNA. In another embodiment, the at least one or more complementary riboregulators (cis) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

[0037] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (cis) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (cis) in the repRNA block the splice acceptor (SA) site and reduce off-target trans-splicing.

[0038] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (trans) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

[0039] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (trans) in the repRNA block the splice acceptor (SA) site and reduce off-target trans splicing.

[0040] In one embodiment, the repRNA contains modifications, the modifications comprising at least one or more binding motifs. In another embodiment, compared to the unmodified form, the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site blocking (SA, SD, ISS, ISE, ESE, and ESS).

[0041] In one embodiment, the repRNA contains modifications that enable it to induce trans-splicing in response to stimuli, compared to its unmodified form. In another embodiment, the repRNA contains modifications that enable it to shut down or reduce trans-splicing in response to stimuli, compared to its unmodified form.

[0042] In one embodiment, the repRNA is modified to enable small molecule-induced trans-splicing compared to its unmodified form. In another embodiment, the repRNA is modified to repress small molecule-induced trans-splicing compared to its unmodified form.

[0043] In the implementation scheme, the repRNA contains modifications that enable it to perform light-induced trans-splicing.

[0044] In the implementation scheme, the repRNA includes modifications, the modifications comprising at least one or more motifs that are bound to and regulated by a light-sensitive protein.

[0045] In an embodiment, the snRNA or snoRNA contains a sequence in the 3' untranslated region (3'UTR). In an embodiment, the sequence in the 3' UTR increases trans-splicing efficiency compared to the unmodified form. In an embodiment, the sequence is derived from the MALAT1 gene. In an embodiment, the sequence is the nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitution, addition, or deletion.

[0046] In one embodiment, the RNP is assembled on the repRNA and / or the target. In another embodiment, the RNP is assembled on the repRNA. In yet another embodiment, the RNP is assembled on the target. In yet another embodiment, the RNP spatially blocks and inhibits cis-splicing.

[0047] In one embodiment, the repRNA comprises a minimal intron. In another embodiment, the minimal intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

[0048] In an embodiment, the repRNA further comprises a guide RNA (gRNA). In an embodiment, the gRNA hybridizes with a target nucleic acid molecule. In an embodiment, the gRNA guides the repRNA to the target nucleic acid molecule. In an embodiment, the guide RNA is or comprises a sequence of SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In the implementation scheme, the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 1... 0, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0049] In embodiments, the compositions or systems disclosed herein comprise a nuclease, wherein the nuclease is a CRISPR-Cas enzyme.

[0050] In one implementation, the Cas is type I. In another implementation, the Cas is type IA, such as, but not limited to, Cas8a or Cas5. In another implementation, the Cas is type I. In another implementation, the Cas is type IB, such as, but not limited to, Cas8b. In another implementation, the Cas is type I. In another implementation, the Cas is type IC, such as, but not limited to, Cas8c. In another implementation, the Cas is type I. In another implementation, the Cas is type ID, such as, but not limited to, Cas10d. In another implementation, the Cas is type I. In another implementation, the Cas is type IE, such as, but not limited to, Cse1 or Cse2. In another implementation, the Cas is type I. In another implementation, the Cas is type IF, such as, but not limited to, Csy1, Csy2, or Csy3. In another implementation, the Cas is type I. In another implementation, the Cas is type IG, such as, but not limited to, GSU0054. In another implementation, the Cas is type I. In another implementation, the Cas type I is, but not limited to, Cas3.

[0051] In one embodiment, the Cas is type II. In another embodiment, the Cas is type II-A, such as, but not limited to, Csn2. In another embodiment, the Cas is type II. In another embodiment, the Cas is type II-B, such as, but not limited to, Cas4. In another embodiment, the Cas is type II. In another embodiment, the Cas is type II-C. In another embodiment, the Cas is type II. In another embodiment, the Cas type II is, but not limited to, Cas 9.

[0052] In one implementation, the Cas is type III. In another implementation, the Cas is type III-A, such as, but not limited to, Csm2. In another implementation, the Cas is type III. In another implementation, the Cas is type III-B, such as, but not limited to, Cmr5. In another implementation, the Cas is type III. In another implementation, the Cas is type III-C, such as, but not limited to, Cas10 or Csx11. In another implementation, the Cas is type III. In another implementation, the Cas is type III-D, such as, but not limited to, Csx10. In another implementation, the Cas is type III. In another implementation, the Cas is type III-E. In another implementation, the Cas is type III. In another implementation, the Cas is type III-F. In another implementation, the Cas is type III. In another implementation, the Cas type III is, but not limited to, Cas 10.

[0053] In one implementation, the Cas is type IV. In another implementation, the Cas is type IV-A. In yet another implementation, the Cas is type IV. In yet another implementation, the Cas is type IV-B. In yet another implementation, the Cas is type IV. In yet another implementation, the Cas is type IV-C.

[0054] In one implementation, the Cas is type V. In another implementation, the Cas is type VA, such as, but not limited to, Cas12a (Cpf1). In another implementation, the Cas is type V. In another implementation, the Cas is type VB, such as, but not limited to, Cas12b (C2c1). In another implementation, the Cas is type V. In another implementation, the Cas is type VC, such as, but not limited to, Cas12c (C2c3). In another implementation, the Cas is type V. In another implementation, the Cas is type VD, such as, but not limited to, Cas12d (CasY). In another implementation, the Cas is type V. In another implementation, the Cas is type VE, such as, but not limited to, Cas12e (CasX). In another implementation, the Cas is type V. In another implementation, the Cas is type VF, such as, but not limited to, Cas12f (Cas14 or C2c10). In another implementation, the Cas is type V. In another implementation, the Cas is type VG, such as, but not limited to, Cas12g. In another implementation, the Cas is type V. In another implementation, the Cas is type VH, such as, but not limited to, Cas12h. In one implementation, the Cas is type V. In another implementation, the Cas is type VI, such as, but not limited to, Cas12i. In another implementation, the Cas is type V. In another implementation, the Cas is type VK, such as, but not limited to, Cas12k (C2c5). In another implementation, the Cas is type V. In another implementation, the Cas is type VU, such as, but not limited to, C2c4, C2c8, or C2c9. In another implementation, the Cas is type V. In another implementation, the Cas V type is, but not limited to, Cas 12. In another implementation, the Cas is type VI.

[0055] In one embodiment, the Cas is type VI-A, such as, but not limited to, Cas13a (C2c2). In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-B, such as, but not limited to, Cas13b. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-C, such as, but not limited to, Cas13c. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-D, such as, but not limited to, Cas13d. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-X, such as, but not limited to, Cas13x.1. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-Y. In another embodiment, the Cas is type VI. In another embodiment, the Cas VI type is, but not limited to, Cas 13.

[0056] In the implementation scheme, the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10 or Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8, C2c9, Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d or Cas13x.1.

[0057] In embodiments, the composition or system comprises a CRISPR-Cas enzyme, such as its protein and / or nucleic acid. In embodiments, the composition or system further comprises repair RNA (repRNA) and a CRISPR-Cas enzyme. In embodiments, the CRISPR / Cas system comprises guide RNA (gRNA) and repRNA in cis or trans configuration, the repRNA being capable of Cas protein binding and trans splicing. In embodiments, the CRISPR-Cas enzyme is active, for example, having catalytic activity. In embodiments, the CRISPR-Cas enzyme is inactive, for example, catalytically inactivated, e.g., "inactivated". In embodiments, the composition or system comprises an intron sequence containing a sequence that interacts with or is suitable for interacting with a CRISPR-Cas enzyme. In embodiments, when the methods of the invention are performed in cis or trans configuration as described herein, the composition or system further comprises repair RNA (repRNA) and a CRISPR-Cas enzyme. In embodiments, the compositions or systems disclosed herein comprise RNA sequences that interact with active or catalytically inactivated endonucleases.

[0058] In one embodiment, the gRNA associates with one or more endonucleases, or is suitable for association with one or more endonucleases. In another embodiment, the endonuclease contains one or more mutations to reduce catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially inactivate the endonuclease catalytically relative to its unmutated form. In yet another embodiment, the endonuclease containing one or more mutations increases catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially overactivate the endonuclease catalytically relative to its unmutated form. In the embodiments, the endonuclease comprises an amino acid sequence or a fragment or variant thereof of one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, and has at least about 70% identity with one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or has about 1 to about 20 amino acid modifications.

[0059] In one embodiment, the repRNA is operatively linked to one or more antisense sequences that bind to the target nucleic acid molecule. In another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In another embodiment, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is provided in a trans configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is operatively linked to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide. In yet another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide.

[0060] In one embodiment, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding peptides. In another embodiment, the repRNA is provided in a trans configuration to one or more sequences that bind to and / or hybridize with RNA-binding peptides. In yet another embodiment, the repRNA is operatively linked to one or more recognition sequences for RNP complex formation.

[0061] In one embodiment, the repRNA is provided in cis configuration to one or more recognition sequences for RNP complex formation. In another embodiment, the repRNA is not operatively linked to one or more recognition sequences for RNP complex formation. In yet another embodiment, the repRNA is provided in trans configuration to one or more recognition sequences for RNP complex formation.

[0062] In one embodiment, the repRNA is operatively linked to one or more gRNAs. In another embodiment, the repRNA is provided cis-associated to one or more gRNAs. In yet another embodiment, the repRNA is not operatively linked to one or more gRNAs. In yet another embodiment, the repRNA is provided trans-associated to one or more gRNAs.

[0063] In some embodiments, the repRNA further comprises a ribozyme site. In some embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis D virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In some embodiments, the ribozyme site is an HDV ribozyme site. In some embodiments, the ribozyme site is a twisted ribozyme site. In some embodiments, the ribozyme site is upstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is downstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme cleaves a target. In some embodiments, the ribozyme is a trans-cleaving ribozyme.

[0064] In embodiments, when the method of the present invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the repRNA contains a ribozyme site that cleaves the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the 3' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 3' end of the repRNA.

[0065] In embodiments, when the method of the invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the snRNA or snoRNA is modified to contain at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence. In embodiments, the snRNA or snoRNA is modified not to contain M6A sites. In embodiments, the repRNA contains at least one or more M6A sites. In one embodiment, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the exon sequence. In another embodiment, the repRNA does not contain M6A sites.

[0066] In one embodiment, the composition or system further comprises at least one precursor rRNA stem-loop. In another embodiment, the at least one precursor rRNA stem-loop has a 5' cap or a 3' polyA tail removed.

[0067] In one embodiment, the repRNA comprises at least one or more snRNA or snoRNA sequences. In another embodiment, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In another embodiment, the repRNA comprises an artificial smU7 system. In another embodiment, the artificial smU7 system stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 5' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 5' end of the snRNA or snoRNA stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 3' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 3' end of the snRNA or snoRNA stabilizes the repRNA.

[0068] In the embodiments, there are multiple repRNAs controlled by the same, different, or multiple promoters. In the embodiments, the repRNAs and one or more other components of the system of the present invention are controlled by the same or different promoters.

[0069] In one embodiment, the repRNA comprises an alternative promoter. In another embodiment, the repRNA comprises at least one or more alternative Pol II promoters. In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine). In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) to stabilize the repRNA.

[0070] In one embodiment, the repRNA comprises at least one or more circularized 5' replacement splice donor (SD) repRNAs. In another embodiment, the at least one or more circularized 5' replacement splice donor (SD) repRNAs stabilize the repRNA. In another embodiment, the repRNA comprising one or more circularized 5' replacement (SD) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In another embodiment, the repRNA comprises at least one or more circularized 3' replacement splice acceptor (SA) repRNAs. In another embodiment, the repRNA comprising at least one or more circularized 3' replacement splice acceptor (SA) repRNAs stabilizes the repRNA. In another embodiment, the repRNA comprising one or more circularized 3' replacement (SA) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In yet another embodiment, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In another embodiment, the at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In the implementation, the repRNA containing one or more circularized internal substitution (SD + SA) repRNAs has improved stability and resistance to exonucleases compared to the unmodified form.

[0071] In embodiments, the composition or system targets or is suitable for targeting one or more Usher syndrome-related genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system targets or is suitable for targeting one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or their precursor mRNA sequences. In embodiments, the composition or system targets or is suitable for targeting one or more genes selected from the following: USH2A, GPR98, and DFNB31, or their precursor mRNA sequences. In embodiments, the composition or system targets or is suitable for targeting USH2A or its precursor mRNA sequence. In embodiments, the composition or system targets or is suitable for targeting exon 13 of USH2A or its precursor mRNA sequence. In embodiments, the composition or system targets or is suitable for replacing c.2299delG and / or c.2276G > T in the USH2A or its precursor mRNA sequence. In embodiments, the composition or system is suitable for correcting mutations or defects in one or more Usher syndrome-related genes.

[0072] In the implementation plan, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

[0073] In embodiments, the composition or system is suitable for correcting mutations or defects in one or more genes selected from: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1 or their precursor mRNA sequences.

[0074] In embodiments, the composition or system is suitable for correcting mutations or defects in one or more genes selected from USH2A, GPR98, and DFNB31, or their precursor mRNA sequences. In embodiments, the composition or system is suitable for correcting mutations or defects in USH2A or its precursor mRNA sequences. In embodiments, the composition or system is suitable for correcting mutations or defects in exon 13 of USH2A or its precursor mRNA sequences. In embodiments, the composition or system is suitable for correcting c.2299delG and / or c.2276G > T in USH2A or its precursor mRNA sequences.

[0075] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0076] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0077] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0078] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0079] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0080] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0081] In one embodiment, the endonuclease is linked, associated with, and / or fused with the RNA-binding protein. In another embodiment, the endonuclease is linked to the RNA-binding protein via a linker. In yet another embodiment, the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.

[0082] In one embodiment, the linker substantially comprises glycine and serine residues. In another embodiment, the linker is (GGS). n ,in n It can be 1, 2, 3, 4, or 5. In the implementation scheme, the connector is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), or (GGGGS). n (n=1-4) (SEQ ID NO: 65) 、 (Gly)8 (SEQ ID NO: 66), (Gly)6 (SEQ ID NO: 67), (EAAAK) n (n=1-3) (SEQ ID NO: 68), A(EAAAK) nA (n = 2-5) (SEQ ID NO: 69), AEAAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75) or variants thereof, wherein the variants contain about 1, about 2, about 3, about 4, or about 5 mutations selected from substitutions or deletions.

[0083] In one embodiment, the repRNA comprises a splicing donor. In another embodiment, the repRNA comprises a splicing acceptor.

[0084] In one embodiment, the repRNA contains exons of the target nucleic acid. In another embodiment, the repRNA contains introns of the target nucleic acid.

[0085] In one embodiment, the repRNA contains one or more non-natural introns. In another embodiment, the target nucleic acid is a precursor mRNA transcript molecule.

[0086] In one embodiment, the target nucleic acid is one or more Usher syndrome-related genes, fragments thereof, or precursor mRNA sequences thereof. In another embodiment, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In another embodiment, the target nucleic acid is one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or precursor mRNA sequences thereof. In another embodiment, the target nucleic acid is one or more genes selected from the following: USH2A, GPR98, and DFNB31, or precursor mRNA sequences thereof. In another embodiment, the target nucleic acid is USH2A or its precursor mRNA sequence. In another embodiment, the target nucleic acid is exon 13 of the USH2A or its precursor mRNA sequence. In the implementation scheme, the target nucleic acid is a USH2A or its precursor mRNA sequence carrying the c.2299delG and / or c.2276G > T mutation.

[0087] In one embodiment, the hybridization is mediated by complete sequence complementarity with one strand of the target nucleic acid molecule. In another embodiment, the hybridization is mediated by partial sequence complementarity with one strand of the target nucleic acid molecule.

[0088] In one embodiment, the repRNA mediates the generation of trans-splicing of corrected and / or wild-type USH2A nucleic acid gene transcripts. In another embodiment, the composition or system substantially prevents or eliminates cis-splicing of nucleic acids.

[0089] In the implementation scheme, the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0090] In the embodiments, the compositions or systems disclosed herein may further comprise a viral vector or a non-viral vector.

[0091] In the implementation scheme, the viral vector is or contains AAV, optionally wherein the AAV is or contains one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

[0092] In embodiments, the compositions or systems disclosed herein further comprise lipid nanoparticles (LNPs), liposomes, lipid complexes, or polymer nanoparticles. In embodiments, the LNPs comprise one or more of ionizable lipids, aminolipids, anionic lipids, neutral lipids, amphiphilic lipids, accessory lipids, structural lipids, PEG lipids, and lipids.

[0093] In some embodiments, the composition or system component is a nucleic acid. In some embodiments, the composition or system component comprises a DNA molecule or an RNA molecule. In some embodiments, the RNA is or comprises mRNA or modified mRNA (mmRNA). In some embodiments, the DNA molecule is or comprises a vector or plasmid. In some embodiments, the nucleic acid comprises a codon-optimized sequence. In some embodiments, the nucleic acid comprises one or more modifications. In some embodiments, the modification is one or more of base modifications and backbone modifications.

[0094] In one embodiment, this document discloses a cell comprising nucleic acids, viral vectors, or lipid nanoparticles of any of the embodiments and / or aspects disclosed herein.

[0095] In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a mammalian cell. In yet another embodiment, the cell is a human cell. In one embodiment, the cell is an immortalized cell. In yet another embodiment, the cell is harvested from a subject.

[0096] In the embodiments, this document discloses a pharmaceutical composition comprising a composition or systemic nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, or a cell of any embodiment and / or aspect disclosed herein, and a pharmaceutically acceptable carrier.

[0097] In one embodiment, this document discloses a kit comprising a container containing a composition or system of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, or a cell of any embodiment and / or aspect disclosed herein, and instructions for trans-splicing the nucleic acid.

[0098] In its implementation, this document discloses a method for targeted trans-splicing of USH2A precursor mRNA in cells, the method comprising contacting cells with a composition or system of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, or a cell of any embodiment and / or aspect disclosed herein.

[0099] In its implementation, this document discloses a method for treating a patient suffering from a disease associated with a mutation in the USH2A gene, the method comprising administering a therapeutically effective amount of a composition or system, a nucleic acid, a viral vector, a lipid nanoparticle, or a cell of any of the embodiments and / or aspects disclosed herein.

[0100] In its implementation, this document discloses a method for treating, improving, or preventing a disease associated with a USH2A gene mutation, the method comprising: contacting cells with a composition or system, a nucleic acid, a viral vector, a lipid nanoparticle, or a cell, any of the embodiments and / or aspects disclosed herein, and administering an effective amount of the cells to a subject.

[0101] In its implementation, this document discloses a method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising administering to the subject an effective amount of a composition or system of any of the embodiments and / or aspects disclosed herein, a nucleic acid of any of the embodiments and / or aspects disclosed herein, a viral vector of any of the embodiments and / or aspects disclosed herein, a lipid nanoparticle of any of the embodiments and / or aspects disclosed herein, or a cell of any of the embodiments and / or aspects disclosed herein.

[0102] In an implementation scheme, this document discloses a method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising: contacting cells with a composition or system as described in any one of claims 1-111, a nucleic acid as described in any one of claims 116-122, a viral vector as described in any one of claims 112-113, a lipid nanoparticle as described in claims 114-115, or a cell as described in any one of claims 123-128, and administering an effective amount of the cells to the subject.

[0103] In the implementation scheme, the cells are derived from the subject.

[0104] In one implementation, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In another implementation, the Usher syndrome is Usher syndrome type I. In yet another implementation, the Usher syndrome is Usher syndrome type II. In yet another implementation, the Usher syndrome is Usher syndrome type III.

[0105] In one embodiment, the method targets one or more Usher syndrome-related genes. In another embodiment, the method targets one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In yet another embodiment, the method targets one or more of USH2A, GPR98, and DFNB31. In yet another embodiment, the method targets USH2A.

[0106] In an implementation, the method corrects mutations or defects in one or more Usher syndrome-related genes. In an implementation, the method corrects mutations or defects in one or more genes selected from the following: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In an implementation, the method corrects mutations or defects in one or more of USH2A, GPR98, and DFNB31. In an implementation, the method corrects mutations or defects in USH2A.

[0107] In one embodiment, the method induces trans splicing of one or more genes selected from the following: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In another embodiment, the method induces trans splicing of one or more of USH2A, GPR98, and DFNB31. In yet another embodiment, the method induces trans splicing of USH2A.

[0108] In the embodiments, the method treats, improves, or prevents one or more symptoms of retinitis pigmentosa. In the embodiments, the method treats, improves, or prevents hearing loss or impairment. In the embodiments, the method treats, improves, or prevents vision loss or impairment. In the embodiments, the method treats, improves, or prevents one or more of night blindness and peripheral vision loss or impairment.

[0109] In various embodiments, the compositions disclosed herein or the systems disclosed herein for targeting nucleic acids to perform trans-splicing include a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing. In embodiments, the trans-splicing system includes a splice donor, a splice acceptor, and a replacement of an inner exon. In embodiments, the repRNA is operatively linked to an RNA molecule or the gRNA comprising a sequence complementary to one strand of a target nucleic acid molecule.

[0110] In several aspects, this disclosure provides a system for targeting nucleic acids for trans-splicing, the system comprising: (a) a nuclease of any embodiment disclosed herein, and an RNA molecule optionally comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide associated with said nuclease; and (c) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; and (ii) a splicing donor and / or splicing acceptor.

[0111] In the implementation scheme, the RNA molecule is gRNA.

[0112] In the implementation scheme, the endonuclease is not linked, associated with, and / or fused with RNA-binding proteins.

[0113] In one embodiment, the repRNA is not operatively linked to one or more gRNAs. In another embodiment, the repRNA is provided trans-formally to one or more gRNAs.

[0114] In some embodiments, the repRNA further comprises a ribozyme site. In some embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis D virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In some embodiments, the ribozyme site is an HDV ribozyme site. In some embodiments, the ribozyme site is a twisted ribozyme site. In some embodiments, the ribozyme site is upstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is downstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme cleaves a target. In some embodiments, the ribozyme is a trans-cleaving ribozyme.

[0115] In embodiments, when the method of the present invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the repRNA contains a ribozyme site that cleaves the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the 3' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 3' end of the repRNA.

[0116] In embodiments, when the method of the invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the snRNA or snoRNA is modified to contain at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence. In embodiments, the snRNA or snoRNA is modified not to contain M6A sites. In embodiments, the repRNA contains at least one or more M6A sites. In one embodiment, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the exon sequence. In another embodiment, the repRNA does not contain M6A sites.

[0117] In one embodiment, the composition or system further comprises at least one precursor rRNA stem-loop. In another embodiment, the at least one precursor rRNA stem-loop has a 5' cap or a 3' polyA tail removed.

[0118] In one embodiment, the repRNA comprises at least one or more snRNA or snoRNA sequences. In another embodiment, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In another embodiment, the repRNA comprises an artificial smU7 system. In another embodiment, the artificial smU7 system stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 5' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 5' end of the snRNA or snoRNA stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 3' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 3' end of the snRNA or snoRNA stabilizes the repRNA.

[0119] In the embodiments, there are multiple repRNAs controlled by the same, different, or multiple promoters. In the embodiments, the repRNAs and one or more other components of the system of the present invention are controlled by the same or different promoters.

[0120] In one embodiment, the repRNA comprises an alternative promoter. In another embodiment, the repRNA comprises at least one or more alternative Pol II promoters. In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine). In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) to stabilize the repRNA.

[0121] In one embodiment, the repRNA comprises at least one or more circularized 5' replacement splice donor (SD) repRNAs. In another embodiment, the at least one or more circularized 5' replacement splice donor (SD) repRNAs stabilize the repRNA. In another embodiment, the repRNA comprising one or more circularized 5' replacement (SD) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In another embodiment, the repRNA comprises at least one or more circularized 3' replacement splice acceptor (SA) repRNAs. In another embodiment, the at least one or more circularized 3' replacement splice acceptor (SA) repRNAs stabilize the repRNA. In another embodiment, the repRNA comprising one or more circularized 3' replacement (SA) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In yet another embodiment, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In another embodiment, the at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In the implementation, the repRNA containing one or more circularized internal substitution (SD+SA) repRNAs has improved stability and resistance to exonucleases compared to the unmodified form.

[0122] In several aspects, this disclosure provides a system for targeting nucleic acids for trans-splicing, the system comprising: (a) a nuclease as described in any one of claims 1-108, and an RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule; and (b) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; and (ii) a splice donor and / or splice acceptor.

[0123] In this embodiment, the RNA molecule is gRNA. In this embodiment, the endonuclease is not linked, associated with, and / or fused with RNA-binding proteins. In this embodiment, the repRNA is operatively linked to one or more gRNAs.

[0124] In an embodiment, the composition comprises gRNA, repRNA, and Cas endonuclease operably linked to a single promoter or a bidirectional promoter.

[0125] In the implementation scheme, the gRNA and repRNA are located on the first side of the bidirectional promoter, and the Cas endonuclease is located on the second side of the bidirectional promoter.

[0126] In several aspects, this disclosure provides a method for slowing down, reducing, or eliminating transcription of target genes and / or stimulating, enhancing, or increasing Pol II arrest / release. In embodiments, without being bound by theory, this method, compared to the unmodified form, increases trans-splicing by forcibly forming a large G4 or similar RNA-RNA motif downstream of the repRNA binding motif.

[0127] In several aspects, this disclosure provides compositions, systems, and / or methods for individually delivering at least one repRNA capable of trans-splicing at least one other repRNA (e.g., tandem) and / or endogenous RNA targets, for example, for performing multi-kilobase editing with a cargo capacity greater than that of a single AAV. In embodiments, this allows for single AAV delivery, for example, by effectively reducing the need for cargo sizes greater than the AAV loading capacity.

[0128] In several aspects, this disclosure provides a method for modifying and converting endogenous RNA (e.g., but not limited to precursor mRNA, mRNA, lncRNA) into repRNA for trans-splicing.

[0129] In an embodiment, the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any of SEQ ID NO: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0130] In one embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids. In another embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids, wherein the USH2A target nucleic acid is selected from exon 13 (SEQ ID NO: 2023) or intron 13 (SEQ ID NO: 2024). In another embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids of SEQ ID NO: 2023 or SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein said fold change is measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2023 binding to any of SEQ ID NO: 804-2022 is greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the multiple change is measured based on the trans-splicing editing rate relative to the non-target rate.

[0131] In the implementation, the binding motif has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation, the binding motif has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5 or 2.5-3.0, said fold change being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2024 binding to any of SEQ ID NO: 804-2022 is greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the multiple change is measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2024 binding to any of SEQ ID NO: 804-2022 is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5 or 2.5-3.0, wherein the fold change is measured based on the trans-splicing editing rate relative to the non-target rate.

[0132] In one embodiment, the binding motif binds to the position of intron 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2024. In yet another embodiment, the binding motif binds to the USH2A target nucleic acid (SEQ ID NO: 2024). The positions of intron 13 of (2024), wherein the positions are selected from approximately positions 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000.In the implementation scheme, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to position 13 of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000.

[0133] In one embodiment, the binding motif binds to the position of intron 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2024. In yet another embodiment, the binding motif binds to the USH2A target nucleic acid (SEQ ID NO: 2024). The position of intron 13 of the target nucleic acid (2024), wherein the position relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, ... 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.In the implementation scheme, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to the USH2A target nucleic acid (SEQ ID NO: 804-2022). The position of intron 13 of (2024) relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450. 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.

[0134] In one embodiment, the binding motif binds to position 13 of exon 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2023. In yet another embodiment, the binding motif binds to position 13 of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80. In yet another embodiment, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to position 13 of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80.

[0135] In one embodiment, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2023. In yet another embodiment, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position relative to the splice donor site is selected from, but not limited to, approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80. In yet another embodiment, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position relative to the splice donor site is selected from, but not limited to, approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80.

[0136] In an embodiment, the binding motif is selected from SEQ ID NO: 804, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, said fold change being measured based on the trans-splicing editing rate relative to the non-target rate. In one embodiment, the binding motif is SEQ ID NO: 804 and binds to a position in intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16, or relative to the splice donor site selected from approximately positions 5 to 20, approximately 8 to 16, or approximately 12 nucleotides from the splice donor site in intron 13 of USH2A. In another embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to a position in intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, approximately 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16. In one embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 12. In another embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, position 12.

[0137] In an embodiment, the binding motif is selected from SEQ ID NO: 804-818, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-818 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130, or relative to the splice donor site selected from approximately positions 5 to 20, approximately 8 to 16, or approximately 12 nucleotides from the splice donor site in intron 13 of USH2A. In one embodiment, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, about 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130. In another embodiment, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from positions 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123. In the implementation scheme, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, positions 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123.

[0138] In an embodiment, the binding motif is selected from SEQ ID NO: 804-883, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-883 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0139] In an embodiment, the binding motif is selected from SEQ ID NO: 804-1019, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-1019 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0140] In an embodiment, the binding motif is selected from SEQ ID NO: 804-1788, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-1788 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0141] In the implementation scheme, the binding motif is selected from SEQ ID NO: 804-1788 and combined to approximately positions 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 10 9, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86, 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 8 4, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69, 7, 2, 8, 102, 92, 36, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104, 126, 20, 36, 9, 27, 35, 138, 121, 170,92、36、890、104、12、870、14、38、34、34、69、72、11、72、16、67、136、143、91、38、1、21、137、83、61、20、36、104、29、8、30、5、84、30、106、78、16、148、9、25、144、87、36、74、44、9、34、14、32、4、6、35、34、76、44、77、73、2、6、2、138、30、850、62、5、137、12、89、39、40、23、79、141、74、30、540、130、14、15、20、23、12、77、146、10、8、2、13、42、35、28、730、24、143、62、4、41、24、37、92、10、840、29、8、45、138、660、38、11、20、39、90、90、107、31、135、37、61、22、2、142、37、4、6、40、87、38、140、38、59、6、29、139、15、9、145、640、15、131、13、141、2、26、18、106、4、32、62、21、14、100、820、60、8、3、33、12、22、7、59、76、47、9、30、59、730、860、26、143、230、540、32、27、54、136、36、17、10、22、36、250、590、155、150、12、105、81、36、8、850、34、35、210、52、840、22、21、18、91、4、820、52、29、28、660、31、63、85、880、18、18、78、890、640、155、34、93、4、81、890、78、28、24、14、22、20、24、70、210、24、147、153、10、34、139、18、19、0、16、470、140、14、220、630、71、26、22、23、15、28、105、93、85、14、420、146、31、8、39、37、35、16、10、530、6、78、21、22、47、60、28、8、77、0、19、78、85、20、660、21、4、45、4、12、5、24、154、31、390、470、4、25、8、7、6、152、140、220、55、1、25、29、4、3、27、14、6、190、45、24、13、46、18、12、46、32、151、410、7、20、350、3、40、620、39、0、880、30、22、155、24、0、157、32、1、8、142、14、440、160、16、64、530、870、147、160、240、17、33、8、2、68、39、33、141、137、76、2、19、400、76、860、58、19、23、8、860、360、7、8、51、820、14、12、1、190、6、26、450、16、150、52、1、47、18、24、27、210、18、57、36、33、650、4、80、70、32、26、16、46、150、29、17、250、600、35、56、60、48、23、37、19、0、154、0、180、27、26、4、830、25、58、149、410、7、152、49、0、160、380、600、16、56、590、0、28、440、58、2、630、53、158、31、2、650、16、240、84、4、57、330、20、16、70、9、62、360、26、26、17、153、50、14、350、2、26、68、52、200、154、18、32、153、27、3、0、53、460、560、600、1、159、16、120、18、0、230、28、730、360、54、1、30、72、105、158、390、390、540、48、880、370、32、17、4、49、36、18、8、500、50、26、2、23、610、25、6、200、145、4、3、450、55、16、80、570、6、460、670、17、152、76、560、16、71、20、74、156、14、7、74、0、220、550、18、15、530、18、350、21、450、400、720、2、66、0、0、630、500、33、14、4、670、6、68、2、370、151、80、50、54、180、7、0、16、560、74、158、22、900、0、1、420、180、31、60、70、270、830、190、280、320、83、78、49、50、51、14、8、420、58、75、650、148、46、70、156、44、900、151、7、720、159、570、370、380、8、720、12、41、270、6、260、200、156、920、52、12、280、33、580、70、0、157、460、72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25, or relative to the splice donor site, bound to approximately position 12, 38, 111, 115, 111, 123, 108, 1 10, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 1 22, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73,94、34、840、6、430、88、27、20、38、99、11、38、129、6、30、10、135、129、69、7、2、8、102、92、36、107、32、135、22、30、28、75、6、41、34、106、146、28、22、71、22、142、32、14、73、44、850、104、126、20、36、9、27、35、138、121、170、92、36、890、104、12、870、14、38、34、34、69、72、11、72、16、67、136、143、91、38、1、21、137、83、61、20、36、104、29、8、30、5、84、30、106、78、16、148、9、25、144、87、36、74、44、9、34、14、32、4、6、35、34、76、44、77、73、2、6、2、138、30、850、62、5、137、12、89、39、40、23、79、141、74、30、540、130、14、15、20、23、12、77、146、10、8、2、13、42、35、28、730、24、143、62、4、41、24、37、92、10、840、29、8、45、138、660、38、11、20、39、90、90、107、31、135、37、61、22、2、142、37、4、6、40、87、38、140、38、59、6、29、139、15、9、145、640、15、131、13、141、2、26、18、106、4、32、62、21、14、100、820、60、8、3、33、12、22、7、59、76、47、9、30、59、730、860、26、143、230、540、32、27、54、136、36、17、10、22、36、250、590、155、150、12、105、81、36、8、850、34、35、210、52、840、22、21、18、91、4、820、52、29、28、660、31、63、85、880、18、18、78、890、640、155、34、93、4、81、890、78、28、24、14、22、20、24、70、210、24、147、153、10、34、139、18、19、0、16、470、140、14、220、630、71、26、22、23、15、28、105、93、85、14、420、146、31、8、39、37、35、16、10、530、6、78、21、22、47、60、28、8、77、0、19、78、85、20、660、21、4、45、4、12、5、24、154、31、390、470、4、25、8、7、6、152、140、220、55、1、25、29、4、3、27、14、6、190、45、24、13、46、18、12、46、32、151、410、7、20、350、3、40、620、39、0、880、30、22、155、24、0、157、32、1、8、142、14、440、160、16、64、530、870、147、160、240、17、33、8、2、68、39、33、141、137、76、2、19、400、76、860、58、19、23、8、860、360、7、8、51、820、14、12、1、190、6、26、450、16、150、52、1、47、18、24、27、210、18、57、36、33、650、4、80、70、32、26、16、46、150、29、17、250、600、35、56、60、48、23、37、19、0、154、0、180、27、26、4、830、25、58、149、410、7、152、49、0、160、380、600、16、56、590、0、28、440、58、2、630、53、158、31、2、650、16、240、84、4、57、330、20、16、70、9、62、360、26、26、17、153、50、14、350、2、26、68、52、200、154、18、32、153、27、3、0、53、460、560、600、1、159、16、120、18、0、230、28、730、360、54、1、30、72、105、158、390、390、540、48、880、370、32、17、4、49、36、18、8、500、50、26、2、23、610、25、6、200、145、4、3、450、55、16、80、570、6、460、670、17、152、76、560、16、71、20、74、156、14、7、74、0、220、550、18、15、530、18、350、21、450、400、720、2、66、0、0、630、500、33、14、4、670、6、68、2、370、151、80、50、54、180、7、0、16、560、74、158、22、900、0、1、420, 180, 31, 60, 70, 270, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151, 7, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0, 157, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25.

[0142] Details of one or more embodiments of this disclosure are set forth in the following description. Other features or advantages of this disclosure will become apparent from the following drawings, the detailed description of several embodiments, and the appended claims. The following description of the drawings illustrates the details of this disclosure. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will become apparent from the specification and claims. In the specification and appended claims, the singular form also includes the plural form unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Attached Figure Description

[0143] Figure 1 This is a non-limiting schematic diagram illustrating the components and assembly of the USH2A trans-splicing system, the splicing editing mechanism, and the trans-splicing products from RNA trans-splicing reporter gene assays.

[0144] Figure 2 The image shows a plasmid containing the target sequence of the USH2A gene, which includes exon 13 and intron 13, and shows the location of the binding motif, thereby allowing the replacement of USH2A exon 13 via 5' trans-splicing.

[0145] Figure 3 The image shows the addition of ribozymes to the target splicing donor in an RNA transsplicing reporter gene assay.

[0146] Figure 4 The figure shows the percentage of trans-splicing editing activity, measured by the percentage of GFP-positive cells, following transient transfection using the USH2A trans-splicing system, which has (i) 5' repRNA (i.e., as used herein, "repRNA" is equivalent to "RNA sequence that binds to an RNA-binding polypeptide") and a reporter gene with a sequence motif having the indicated binding motif (first bar from left in each group on the x-axis); (ii) 5' repRNA with the HDV ribozyme and a reporter gene with a sequence motif having the indicated binding motif (second bar from left in each group on the x-axis); or (iii) repRNA with only the sequence motif having the indicated binding motif (third bar from left in each group on the x-axis).

[0147] Figure 5 The figure shows the percentage of transsplicing activity, measured by the percentage of GFP-positive cells, following integration transfection using the USH2A transsplicing system, which comprises (i) a reporter gene; (ii) a repRNA with a binding motif; (iii) a Cas protein; and / or (iv) a Cas gRNA. Figure 5 In this context, "NT" refers to a randomized, non-targeted polynucleotide control (SEQ ID NO: 138). Figure 5 In the second row, the numbers 2 and 5 refer to binding motifs (SEQ ID NO: 132 and 135, respectively), and the numbers 1, 2 and 3 in the last row refer to Cas guide RNA (SEQ ID NO: 139-141, respectively).

[0148] Figure 6 This is a graphic illustrating 5' editing applied to the USH2A reporter gene, where gRNA targeting intron 12 is contrasted with a non-targeting guide, the latter's activity of which is driven by the presence of repair RNA. Figure 6 In this context, "NT" refers to a random non-targeted polynucleotide control. The endonuclease repeat sequence used to formulate the guide RNA is SEQ ID NO: 30.

[0149] Figure 7A , Figure 7B , Figure 7C and Figure 7DImages showing the protein sizes of the Cas13K2F system for SEQ ID NO: 1 (Cas13K2F1), SEQ ID NO: 2 (Cas13K2F2), SEQ ID NO: 3 (Cas13K2F3), SEQ ID NO: 4 (Cas13K2F5), SEQ ID NO: 80 (Cas13K2F7), SEQ ID NO: 81 (Cas13K2F8), SEQ ID NO: 82 (Cas13K2F9), SEQ ID NO: 83 (Cas13K2F10), SEQ ID NO: 84 (Cas13K2F11), SEQ ID NO: 85 (Cas13K2F12), SEQ ID NO: 86 (Cas13K2F13), and SEQ ID NO: 87 (Cas13K2F14). Figure 7A , Figure 7B , Figure 7C and Figure 7D The red or black arrows indicate the placement of the nucleotide-binding (HEPN) domain in each protein of higher eukaryotes and prokaryotes.

[0150] Figure 8This is a matrix of identity percentages for the following sequences: SEQ ID NO: 6 (Cas13X.1), SEQ ID NO: 7 (Cas13bt3), SEQ ID NO: 8 (Cas13bt2), SEQ ID NO: 9 (Cas13bt1), SEQ ID NO: 10 (Cas13bt8), SEQ ID NO: 11 (Cas13X.2), SEQ ID NO: 12 (Cas13bt9), SEQ ID NO: 13 (Cas13bt11), SEQ ID NO: 14 (Cas13bt5), SEQ ID NO: 15 (Cas13bt10), SEQ ID NO: 16 (Cas13bt15), SEQ ID NO: 17 (Cas13bt7), SEQ ID NO: 18 (Cas13bt6), SEQ ID NO: 19 (Cas13bt14), SEQ ID NO: 20 (Cas13Y.3), SEQ ID NO: 21. (Cas13bt12), SEQ ID NO: 22 (Cas13Y.1), SEQ ID NO: 23 (Cas13bt4), SEQ ID NO: 24 (Cas13bt16), SEQ ID NO: 25 (Cas13Y.5) and SEQ ID NO: 26 (Cas13Y.4).

[0151] Figure 9Images showing the maximum likelihood phylogenetic tree of the following sequences are provided: SEQ ID NO: 6 (Cas13X.1), SEQ ID NO: 7 (Cas13bt3), SEQ ID NO: 8 (Cas13bt2), SEQ ID NO: 9 (Cas13bt1), SEQ ID NO: 10 (Cas13bt8), SEQ ID NO: 11 (Cas13X.2), SEQ ID NO: 12 (Cas13bt9), SEQ ID NO: 13 (Cas13bt11), SEQ ID NO: 14 (Cas13bt5), SEQ ID NO: 15 (Cas13bt10), SEQ ID NO: 16 (Cas13bt15), SEQ ID NO: 17 (Cas13bt7), SEQ ID NO: 18 (Cas13bt6), SEQ ID NO: 19 (Cas13bt14), SEQ ID NO: 20 (Cas13Y.3), SEQ ID NO: 21 (Cas13bt12), SEQ ID NO: 22 (Cas13Y.1), SEQ ID NO: 23 (Cas13bt4), SEQ ID NO: 24 (Cas13bt16), SEQ ID NO: 25 (Cas13Y.5) and SEQ ID NO: 26 (Cas13Y.4).

[0152] Figure 10 The figure shows a set of binding motif (BM) sequences (i.e., SEQ ID NO: 804-2022) in the repair RNA (repRNA) and screening data on their ability to target USH2A. Detailed Implementation

[0153] This disclosure provides, in particular, compositions, systems, and methods relating to compositions comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splicing donor and / or splicing acceptor, wherein the repRNA is adapted for trans-splicing.

[0154] This disclosure is based in part on the discovery of compositions comprising a composition containing a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing, and methods of using thereof.

[0155] This disclosure is also based in part on the discovery of a system for trans-splicing a target nucleic acid containing a repRNA, the repRNA comprising: (a) one or more exons and / or introns of a target nucleic acid molecule; and (b) a splicing donor and / or splicing acceptor, and a method of using the system.

[0156] Usher syndrome

[0157] Usher syndrome is an autosomal recessive genetic disorder characterized by hearing loss or deafness and progressive vision loss. The vision loss is caused by retinitis pigmentosa (RP), which affects the photoreceptor layer of the retina. However, it is not strictly necessary to be bound by theory; vision loss occurs due to the gradual degeneration of retinal photoreceptor cells.

[0158] Three main types of Usher syndrome have been identified, designated as type I (subtypes IA to IG), type II (subtypes IIA, IIB, and IIC), and type III. These types are distinguished based on their severity and the age at which signs and symptoms appear.

[0159] Subjects with Usher syndrome type I were born with severe hearing loss and began to lose their sight in the first ten years of their lives. Due to problems with their vestibular system, they also exhibited balance difficulties and slow walking as children.

[0160] Usher syndrome type II is a heterogeneous autosomal recessive disorder characterized by progressive retinitis pigmentosa and sensorineural hearing loss, leading to deafness and blindness. Individuals with Usher II typically experience hearing loss rather than deafness, and their hearing does not deteriorate over time. Their balance appears to be normal. They usually begin to lose vision in their second decade of life but may retain some vision by middle age.

[0161] Subjects with Usher syndrome III experience progressive hearing loss, and about half develop balance difficulties. CLRN1 mutations are associated with Usher syndrome type III. CLRN1 encodes clarin-1, a protein involved in the development and maintenance of the inner ear and retina.

[0162] Several genes are associated with or related to Usher syndrome, including mutations in the following genes: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. Mutations in any of these genes can alter gene expression or protein function and may contribute to Usher syndrome. These genes play a role in the development and maintenance of hair cells, which have sensory functions in the inner ear and help transmit sound and motion signals to the brain. In the retina, these genes are also involved in determining the structure and function of photoreceptor cells (called rods and cones). In some cases, the exact role of these genes in hearing and vision is unclear. Most mutations that lead to Usher syndrome result in the loss or reduction of inner ear hair cells, as well as the gradual loss or reduction of retinal rods and cones. The degeneration of these sensory cells leads to the hearing loss, balance problems, and vision loss characteristic of this disorder.

[0163] Usher syndrome type II may be caused by mutations in genes such as USH2A, ADDRV1, WHRN, GPR98 (also known as VLGR1), and DFNB31. Usher syndrome type III may be caused by mutations in genes such as CLRN1.

[0164] The USH2A gene encodes the protein usherin, located in the supporting tissues of the inner ear and retina. Usherin is involved in the development and maintenance of these structures. WHRN mutations are associated with Usher syndrome type 2D or non-syndromic hearing loss (DFNB31). The associated phenotype depends on the location of the mutation within the two main expressed variants (long and short). Variant ADGRV1 is the cause of Usher syndrome type 2, and the associated phenotype is unclear.

[0165] In embodiments, this disclosure provides a composition comprising a repRNA sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing. In embodiments, this disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In embodiments, the composition or system targets or is adapted to target one or more Usher syndrome-related genes. In embodiments, the composition or system targets or is adapted to target one or more Usher syndrome-related genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system targets or is suitable for targeting one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or their precursor mRNA sequences. In embodiments, the composition or system targets or is suitable for targeting one or more genes selected from the following: USH2A, GPR98, and DFNB31, or their precursor mRNA sequences.

[0166] In an embodiment, the composition or system targets or is suitable for targeting the USH2A or its precursor mRNA sequence. In an embodiment, the composition or system targets or is suitable for targeting exon 13 of the USH2A or its precursor mRNA sequence.

[0167] In an embodiment, the composition or system targets or is suitable for targeting c.2299delG and / or c.2276G > T of the USH2A or its precursor mRNA sequence.

[0168] In embodiments, the composition or system is suitable for correcting mutations or defects in one or more Usher syndrome-related genes. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the composition or system is suitable for correcting mutations or defects in one or more genes selected from: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or their precursor mRNA sequences. In embodiments, the composition or system is suitable for correcting mutations or defects in one or more genes selected from: USH2A, GPR98, and DFNB31, or their precursor mRNA sequences. In embodiments, the composition or system is suitable for correcting mutations or defects in USH2A or its precursor mRNA sequence. In embodiments, the composition or system is suitable for correcting mutations or defects in exon 13 of USH2A or its precursor mRNA sequence. In an embodiment, the composition or system is suitable for correcting c.2299delG and / or c.2276G > T in the USH2A or its precursor mRNA sequence.

[0169] In embodiments, the target nucleic acid of the compositions or systems disclosed herein is one or more Usher syndrome-related genes or fragments thereof or precursor mRNA sequences thereof. In embodiments, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In embodiments, the target nucleic acid of the compositions or systems disclosed herein is one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1 or precursor mRNA sequences thereof. In embodiments, the target nucleic acid is one or more genes selected from the following: USH2A, GPR98, and DFNB31 or precursor mRNA sequences thereof. In embodiments, the target nucleic acid is USH2A or its precursor mRNA sequence. In embodiments, the target nucleic acid is exon 13 of the USH2A or its precursor mRNA sequence. In the implementation scheme, the target nucleic acid is a USH2A or its precursor mRNA sequence carrying the c.2299delG and / or c.2276G > T mutation.

[0170] In an embodiment, the composition containing the repRNA sequence or the system for trans-splicing a target nucleic acid containing the repRNA is for treating a disease, such as Usher syndrome type II.

[0171] In its implementation, this document discloses a method for targeted trans-splicing of USH2A precursor mRNA in cells, the method comprising contacting cells with a composition or system of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, or a cell of any embodiment and / or aspect disclosed herein.

[0172] In an embodiment, this disclosure provides a method for treating a patient suffering from a disease associated with a USH2A gene mutation, the method comprising administering a therapeutically effective amount of a composition or system of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, or a cell of any embodiment and / or aspect disclosed herein.

[0173] In its implementation, this document discloses a method for treating, improving, or preventing a disease associated with a USH2A gene mutation, the method comprising: contacting cells with a composition or system, a nucleic acid, a viral vector, a lipid nanoparticle, or a cell, any of the embodiments and / or aspects disclosed herein, and administering an effective amount of the cells to a subject.

[0174] In its implementation, this disclosure provides a method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising administering to the subject an effective amount of a composition or system of any of the embodiments and / or aspects disclosed herein, a nucleic acid of any of the embodiments and / or aspects disclosed herein, a viral vector of any of the embodiments and / or aspects disclosed herein, a lipid nanoparticle of any of the embodiments and / or aspects disclosed herein, or a cell of any of the embodiments and / or aspects disclosed herein.

[0175] In an embodiment, this disclosure provides a method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising: (a) contacting cells with a composition or system, a nucleic acid, a viral vector, a lipid nanoparticle, or a cell as disclosed herein, according to any embodiment and / or aspect thereof; and (b) administering an effective amount of the cells to the subject.

[0176] In the implementation scheme, the cells are derived from the subject.

[0177] In one implementation, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In another implementation, the Usher syndrome is Usher syndrome type I. In yet another implementation, the Usher syndrome is Usher syndrome type II. In yet another implementation, the Usher syndrome is Usher syndrome type III.

[0178] In one embodiment, the method targets one or more Usher syndrome-related genes. In another embodiment, the method targets one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In yet another embodiment, the method targets one or more of USH2A, GPR98, and DFNB31. In yet another embodiment, the method targets USH2A.

[0179] In the implementation scheme, the method corrects mutations or defects in one or more Usher syndrome-related genes.

[0180] In an embodiment, the method corrects mutations or defects in one or more genes selected from the following: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In an embodiment, the method corrects mutations or defects in one or more of USH2A, GPR98, and DFNB31. In an embodiment, the method corrects mutations or defects in USH2A.

[0181] In one embodiment, the method induces trans splicing of one or more genes selected from the following: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1. In another embodiment, the method induces trans splicing of one or more of USH2A, GPR98, and DFNB31. In yet another embodiment, the method induces trans splicing of USH2A.

[0182] In the implementation plan, the method treats, improves, or prevents hearing loss or deafness.

[0183] In the implementation plan, the method treats, improves, or prevents vision loss or impairment.

[0184] In the implementation plan, the method treats, improves, or prevents one or more of night blindness and peripheral vision loss or decline.

[0185] In various implementations, "subject" refers to any animal (e.g., a mammal), including but not limited to humans and non-human animals (including but not limited to non-human primates, dogs, cats, rodents, horses, cattle, pigs, mice, rats, hamsters, rabbits, etc., which will become recipients of a specific treatment or from which cells are harvested)). In some implementations, the subject is a human.

[0186] Repair RNA and guide RNA

[0187] This disclosure provides, in several aspects, a composition comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing. Additionally, this disclosure provides, in several aspects, a system for trans-splicing a target nucleic acid comprising repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In embodiments, the one or more exons are or comprise one or more exons of a target nucleic acid molecule. In embodiments, the one or more introns are or comprise one or more introns of a target nucleic acid molecule. In embodiments, the one or more exons are or comprise one or more exons of the target nucleic acid molecule. In embodiments, the one or more introns are or comprise one or more introns of the target nucleic acid molecule.

[0188] In an embodiment, the repRNA further comprises a guide RNA (gRNA). In an embodiment, the gRNA hybridizes with a target nucleic acid molecule. In an embodiment, the gRNA guides the repRNA to the target nucleic acid molecule. In an embodiment, the guide RNA is or comprises a sequence of SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In the implementation scheme, the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 1... 0, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0189] In one embodiment, the gRNA associates with one or more endonucleases, or is suitable for association with one or more endonucleases. In another embodiment, the endonuclease contains one or more mutations to reduce catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially inactivate the endonuclease catalytically relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations. In yet another embodiment, the one or more mutations increase catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially overactivate the endonuclease catalytically relative to its unmutated form. In the embodiments, the endonuclease comprises an amino acid sequence or a fragment or variant thereof of one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, and has at least about 70% identity with one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or has about 1 to about 20 amino acid modifications.

[0190] In one embodiment, the repRNA is operatively linked to one or more antisense sequences that bind to the target nucleic acid molecule. In another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In another embodiment, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is provided in a trans configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is operatively linked to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide. In yet another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide.

[0191] In one embodiment, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding peptides. In another embodiment, the repRNA is provided in a trans configuration to one or more sequences that bind to and / or hybridize with RNA-binding peptides. In yet another embodiment, the repRNA is operatively linked to one or more recognition sequences for RNP complex formation.

[0192] In one embodiment, the repRNA is provided in cis configuration to one or more recognition sequences for RNP complex formation. In another embodiment, the repRNA is not operatively linked to one or more recognition sequences for RNP complex formation. In yet another embodiment, the repRNA is provided in trans configuration to one or more recognition sequences for RNP complex formation.

[0193] In one embodiment, the repRNA is operatively linked to one or more gRNAs. In another embodiment, the repRNA is provided cis-associated to one or more gRNAs. In yet another embodiment, the repRNA is not operatively linked to one or more gRNAs. In yet another embodiment, the repRNA is provided trans-associated to one or more gRNAs.

[0194] In an implementation, the one or more binding motifs include a recognition sequence for the formation of the RNP complex.

[0195] In embodiments, the composition or system further comprises a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to the snRNA or snoRNA), or a nucleic acid encoding the protein forming or within the RNP. In embodiments, the one or more binding motifs (e.g., but not limited to snRNA or snoRNA), the protein forming or within the RNP, the protein within the RNP, and / or the nucleic acid encoding the protein forming or within the RNP contains modifications or mutations that attenuate, weaken, reduce, decrease, or eliminate RNP activity compared to an unmodified form, and / or result in attenuated RNA modification activity, the RNP activity optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation. In one embodiment, the one or more binding motifs (e.g., but not limited to snRNA or snoRNA), the protein forming or within the RNP, and / or the nucleic acid encoding the protein forming or within the RNP contains modifications or mutations that increase, stimulate, or enhance RNP activity, or enhance RNA modification activity, compared to the unmodified form, wherein the RNP activity is optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation, compared to the unmodified form. In another embodiment, the snRNA, snoRNA, the protein forming or within the RNP, and / or the nucleic acid encoding the protein forming or within the RNP contains at least one or more pseudouridineization sites. In yet another embodiment, the snRNA, snoRNA, the protein forming or within the RNP, and / or the nucleic acid encoding the protein forming or within the RNP does not contain pseudouridineization sites. In yet another embodiment, the repRNA contains at least one or more pseudouridineization sites. In yet another embodiment, the repRNA does not contain pseudouridineization sites. In the implementation scheme, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more pseudouridine sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of pseudouridine sites in the exon sequence.

[0196] In embodiments, the composition or system comprises repair RNA (repRNA) and / or a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to snRNA or snoRNA), or nucleic acid encoding the protein forming or within the RNP, and / or a small RNA inducing cleavage of RNA and / or CRISPR-Cas enzymes. In embodiments, when the method of the invention is performed in cis or trans as described herein, the composition or system comprises repair RNA (repRNA) and / or a protein forming or within an RNP, the protein having one or more binding motifs (e.g., but not limited to snRNA or snoRNA), or nucleic acid encoding the protein forming or within the RNP, and / or a small RNA inducing cleavage of RNA and / or CRISPR-Cas enzymes. In embodiments, cleavage is initiated by an RNP formed on the repRNA, or by an RNP formed in cis or trans.

[0197] In an embodiment, the one or more binding motifs comprise sequences from small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises a sequence from the snRNA or the snoRNA. In an embodiment, the snoRNA comprises or is an H / ACA snoRNA. In an embodiment, the snoRNA comprises or is a C / D snoRNA.

[0198] In the implementation scheme, the snRNA or snoRNA targets one or more exon splice enhancers (ESEs), one or more intron splice enhancers (ISEs), one or more exon splice silencers (ESSs), and / or one or more intron splice silencers (ISSs). In the implementation scheme, the snRNA or snoRNA is modified to include at least one or more exon splice enhancers (ESEs), at least one or more intron splice enhancers (ISEs), at least one or more exon splice silencers (ESSs), and / or at least one or more intron splice silencers (ISSs).

[0199] In embodiments, the composition or system comprises repair RNA (repRNA) and a small RNA that induces RNA cleavage. In embodiments, the small RNA that induces RNA cleavage is one or more of the following: siRNA, small hairpin RNA (shRNA), U7 snRNA, U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and antisense oligonucleotides (ASO). In embodiments, the composition or system comprises repair RNA (repRNA), and the small RNA contains a modification or mutation that attenuates, weakens, reduces, diminishes, or eliminates activity compared to the unmodified form. In embodiments, the composition or system comprises repair RNA (repRNA), and the small RNA contains a modification or mutation that increases, stimulates, or enhances activity compared to the unmodified form. In embodiments, when the method of the invention is performed in cis or trans as described herein, the composition or system comprises repair RNA (repRNA) and a small RNA that induces RNA cleavage.

[0200] In the implementation scheme, the snRNA or snoRNA contains N 6 -Methyladenosine (M6A) modification. In embodiments, when the method of the invention is performed in cis or trans configuration as described herein, the snRNA contains M6A modification. In embodiments, the snRNA or snoRNA is modified to contain at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence. In embodiments, the snRNA or snoRNA is modified not to contain M6A sites. In embodiments, the repRNA contains at least one or more M6A sites. In one embodiment, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the exon sequence. In another embodiment, the repRNA does not contain M6A sites.

[0201] In one embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence increases the trans-splicing efficiency of the target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In yet another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence decreases the trans-splicing efficiency of the target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In yet another embodiment, compared to the unmodified form, the at least one intronic spacer sequence comprising at least one ISE and ESS sequence increases the trans-splicing efficiency of off-target RNA. In yet another embodiment, the repRNA comprises at least one intronic spacer sequence, the intronic spacer sequence comprising at least one ISE and ESS sequence. In the implementation, compared to the unmodified form, the at least one intron spacer sequence containing at least one ISE and ESS sequence reduces the efficiency of off-target RNA trans-splicing.

[0202] In one embodiment, the repRNA comprises ESS, ESE, ISS, and / or ISE sequences. In another embodiment, the repRNA targets one or more of ESS, ESE, ISS, and / or ISE. In another embodiment, interactions, regulation, and / or binding with one or more of ESS, ESE, ISS, and / or ISE reduce or eliminate the interaction, regulation, and / or binding of one or more of ESS, ESE, ISS, and / or ISE to the target. In another embodiment, the repRNA comprises exon sequences having ESE and ESS sequences. In another embodiment, the exon sequences having ESE and ESS sequences increase or decrease trans-splicing efficiency against the RNA target compared to the unmodified form. In another embodiment, the repRNA comprises exon sequences having ESE and ESS sequences. In another embodiment, the repRNA comprising exon sequences having ESE and ESS sequences increases or decreases trans-splicing efficiency against RNA off-target effects compared to the unmodified form. In another embodiment, the repRNA comprises at least one or more G4 structures. In one embodiment, the repRNA comprises at least one or more G4 structures that isolate SD / SA motifs. In another embodiment, the G4 structure is unwound, for example by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., an endogenous target or exogenously delivered trigger RNA). In this embodiment, the G4 structure reduces off-target effects compared to the unmodified form.

[0203] In one embodiment, the repRNA includes a modification comprising at least one or more scaffold sequences. In another embodiment, the at least one or more scaffold sequences mediate (e.g., recruit) condensate-like aggregation and / or improve the local concentration of the repRNA compared to the unmodified form and other target proteins and / or RNAs. In yet another embodiment, the repRNA includes a modification comprising at least one or more sequences to target the repRNA to the promoter of a target gene of interest, or to a proximal condensate that may contain the promoter. In yet another embodiment, the one or more sequences comprise enhancer RNA, snRNA, and / or snoRNA sequences.

[0204] In one embodiment, the repRNA contains a modification. In another embodiment, the modification improves the interaction and localization with the non-template strand DNA sequence of the target gene compared to the unmodified form. In another embodiment, the non-template strand DNA sequence of the target gene is a promoter, intron, exon, or enhancer. In yet another embodiment, the modification improves the interaction and localization with the non-template strand DNA sequence of the target gene via a protein-directed (e.g., transcription factor, dCas, ZNF, or other RBP) or nucleotide-directed (e.g., R-loop) approach compared to the unmodified form.

[0205] In one embodiment, the repRNA includes a modification comprising an additional RNA element. In another embodiment, the additional RNA element improves subnuclear localization to nuclear spots compared to the unmodified form, thereby enhancing trans-splicing efficiency. In another embodiment, the additional RNA element comprises NEAT1 and / or MALAT1 or fragments thereof. In another embodiment, the additional RNA element comprises the nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitution, addition, or deletion. In another embodiment, the repRNA includes a modification enabling it to target the transcription site of the target RNA. In another embodiment, the repRNA includes a modification comprising a 5' UTR or a 3' UTR modification. In the implementation, the modification containing 5' UTR or 3' UTR alters intracellular or nuclear localization based on interaction with endogenously or exogenously supplied molecules (e.g., the interaction of RNA G4 with transcription factors or other proteins localized to specific cellular compartments).

[0206] In one embodiment, the repRNA contains a modification in its 5' UTR. In another embodiment, the modification in the 5' UTR of the repRNA increases stability compared to the unmodified form. In yet another embodiment, the modification in the 5' UTR of the repRNA decreases stability compared to the unmodified form. In yet another embodiment, the modification in the 5' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form. In another embodiment, the repRNA contains a modification in its 3' UTR. In yet another embodiment, the modification in the 3' UTR of the repRNA increases stability compared to the unmodified form. In yet another embodiment, the modification in the 3' UTR of the repRNA decreases stability compared to the unmodified form. In yet another embodiment, the modification in the 3' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form.

[0207] In an implementation scheme, the repRNA includes a modification comprising modifying the repRNA to include a G4 structure, the G4 structure mediating the recruitment of splice-related RBPs.

[0208] In an embodiment, the repRNA includes modifications that include at least one or more foothold switches in the repRNA. In an embodiment, the at least one or more foothold switches in the repRNA are conditionally activated or deactivated (e.g., SD / SA blocking, binding motif blocking, or RBP blocking) upon detection of endogenously or exogenously supplied target RNA.

[0209] In one embodiment, the repRNA includes a modification comprising at least one or more complementary riboregulators (cis) in the repRNA. In another embodiment, the at least one or more complementary riboregulators (cis) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

[0210] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (cis) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (cis) in the repRNA block the splice acceptor (SA) site and reduce off-target trans-splicing.

[0211] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (trans) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

[0212] In one embodiment, the repRNA includes a modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA. In another embodiment, the at least one or more self-complementary riboregulators (trans) in the repRNA block the splice acceptor (SA) site and reduce off-target trans splicing.

[0213] In one embodiment, the repRNA contains modifications, the modifications comprising at least one or more binding motifs. In another embodiment, compared to the unmodified form, the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site blocking (SA, SD, ISS, ISE, ESE, and ESS).

[0214] In one embodiment, the repRNA contains modifications that enable it to induce trans-splicing in response to stimuli, compared to its unmodified form. In another embodiment, the repRNA contains modifications that enable it to shut down or reduce trans-splicing in response to stimuli, compared to its unmodified form.

[0215] In one embodiment, the repRNA is modified to enable small molecule-induced trans-splicing compared to its unmodified form. In another embodiment, the repRNA is modified to repress small molecule-induced trans-splicing compared to its unmodified form.

[0216] In the implementation scheme, the repRNA contains modifications that enable it to perform light-induced trans-splicing.

[0217] In the implementation scheme, the repRNA includes modifications, the modifications comprising at least one or more motifs that are bound to and regulated by a light-sensitive protein.

[0218] In an embodiment, the snRNA or snoRNA contains a sequence in the 3' untranslated region (3'UTR). In an embodiment, the sequence in the 3' UTR increases trans-splicing efficiency compared to the unmodified form. In an embodiment, the sequence is derived from the MALAT1 gene. In an embodiment, the sequence is the nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, optionally selected from substitution, addition, or deletion.

[0219] In one embodiment, the RNP is assembled on the repRNA and / or the target. In another embodiment, the RNP is assembled on the repRNA. In yet another embodiment, the RNP is assembled on the target. In yet another embodiment, the RNP spatially blocks and inhibits cis-splicing.

[0220] In one embodiment, the repRNA comprises a minimal intron. In another embodiment, the minimal intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

[0221] In an embodiment, the snRNA comprises U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 or U11, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition or deletion. In the embodiments, the snRNA or snoRNA is selected from any of SEQ ID NO: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion, optionally wherein any of SEQ ID NO: 144-802, or a fragment or variant thereof, forms an RNP complex, said fragment or variant optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0222] In some embodiments, the repRNA further comprises a ribozyme site. In some embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis D virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof. In some embodiments, the ribozyme site is an HDV ribozyme site. In some embodiments, the ribozyme site is a twisted ribozyme site. In some embodiments, the ribozyme site is upstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is downstream of one or more exons and / or introns of the repRNA. In some embodiments, the ribozyme site is upstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme site is downstream of the splice donor and / or splice acceptor of the repRNA. In some embodiments, the ribozyme cleaves a target. In some embodiments, the ribozyme is a trans-cleaving ribozyme.

[0223] In embodiments, when the method of the present invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the repRNA contains a ribozyme site that cleaves the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the 3' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 5' end of the repRNA. In embodiments, the repRNA contains a ribozyme site that cleaves the snRNA or snoRNA at the 3' end of the repRNA.

[0224] In embodiments, when the method of the invention is performed in cis or trans configuration as described herein, the repRNA contains M6A modification. In embodiments, the snRNA or snoRNA is modified to contain at least one or more M6A sites. In embodiments, the snRNA or snoRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence. In embodiments, the snRNA or snoRNA is modified not to contain M6A sites. In embodiments, the repRNA contains at least one or more M6A sites. In one embodiment, the repRNA is modified to contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the exon sequence. In another embodiment, the repRNA does not contain M6A sites.

[0225] In one embodiment, the composition or system further comprises at least one precursor rRNA stem-loop. In another embodiment, the at least one precursor rRNA stem-loop has a 5' cap or a 3' polyA tail removed.

[0226] In one embodiment, the repRNA comprises at least one or more snRNA or snoRNA sequences. In another embodiment, the at least one or more snRNA or snoRNA sequences stabilize the repRNA. In another embodiment, the repRNA comprises an artificial smU7 system. In another embodiment, the artificial smU7 system stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 5' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 5' end of the snRNA or snoRNA stabilizes the repRNA. In another embodiment, the at least one or more snRNA or snoRNA sequences include a pseudo-tangle at the 3' end of the snRNA or snoRNA. In another embodiment, the pseudo-tangle at the 3' end of the snRNA or snoRNA stabilizes the repRNA.

[0227] In the embodiments, there are multiple repRNAs controlled by the same, different, or multiple promoters. In the embodiments, the repRNAs and one or more other components of the system of the present invention are controlled by the same or different promoters.

[0228] In one embodiment, the repRNA comprises an alternative promoter. In another embodiment, the repRNA comprises at least one or more alternative Pol II promoters. In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine). In yet another embodiment, the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) to stabilize the repRNA.

[0229] In one embodiment, the repRNA comprises at least one or more circularized 5' replacement splice donor (SD) repRNAs. In another embodiment, the at least one or more circularized 5' replacement splice donor (SD) repRNAs stabilize the repRNA. In another embodiment, the repRNA comprising one or more circularized 5' replacement (SD) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In another embodiment, the repRNA comprises at least one or more circularized 3' replacement splice acceptor (SA) repRNAs. In another embodiment, the at least one or more circularized 3' replacement splice acceptor (SA) repRNAs stabilize the repRNA. In another embodiment, the repRNA comprising one or more circularized 3' replacement (SA) repRNAs has improved stability and is resistant to exonucleases compared to the unmodified form. In yet another embodiment, the repRNA comprises at least one or more circularized internal replacement (SD + SA) repRNAs. In another embodiment, at least one or more circularized internal replacement (SD + SA) repRNAs stabilize the repRNA. In the implementation, the repRNA containing one or more circularized internal substitution (SD+SA) repRNAs has improved stability and resistance to exonucleases compared to the unmodified form.

[0230] How to use

[0231] In several aspects, this disclosure provides a method for modifying nucleic acids in cells using an exon skipping approach, the method comprising contacting cells with a composition of any embodiment and / or aspect of the present disclosure, a nucleic acid of any embodiment and / or aspect of the present disclosure, a viral vector of any embodiment and / or aspect of the present disclosure, a lipid nanoparticle of any embodiment and / or aspect of the present disclosure, a cell of any embodiment and / or aspect of the present disclosure, or a pharmaceutical composition of any embodiment and / or aspect of the present disclosure.

[0232] In several aspects, this disclosure provides a method for modifying nucleic acids in the cells of a subject in need using an exon skipping approach, the method comprising administering an effective amount of a composition of any of the embodiments and / or aspects disclosed herein, a nucleic acid of any of the embodiments and / or aspects disclosed herein, a viral vector of any of the embodiments and / or aspects disclosed herein, a lipid nanoparticle of any of the embodiments and / or aspects disclosed herein, a cell of any of the embodiments and / or aspects disclosed herein, or a pharmaceutical composition of any of the embodiments and / or aspects disclosed herein.

[0233] In one embodiment, the cell is induced to skip erroneous portions of the precursor mRNA molecule by interfering with mRNA splicing. In another embodiment, the method produces a truncated but functional protein, albeit with mutations. In yet another embodiment, the exon skipping method involves binding an oligonucleotide (e.g., but not limited to the RNA molecule of the present invention) to a splicing site in the precursor mRNA molecule. In yet another embodiment, when the oligonucleotide-bound precursor mRNA is processed into mature mRNA, the corresponding exon is skipped, for example, thereby restoring the disrupted reading frame caused by the mutation. In yet another embodiment, exon skipping allows the translation of a protein that is internally missing but substantially functional.

[0234] In embodiments, the exon skipping method of the present invention includes generating single-strand or double-strand breaks in a gene. In embodiments, the single-strand or double-strand breaks result in persistently altered splicing of the gene. In embodiments, the altered splicing results in the expression of a truncated protein, which at least lacks a polypeptide sequence corresponding to the mutated exon. In embodiments, the single-strand or double-strand breaks remove a splice acceptor site or generate a nonfunctional splice acceptor site in or near an exon of the gene, or remove a splice donor site or generate a nonfunctional splice donor site in or near an exon of the gene.

[0235] In several aspects, this disclosure provides for the use of compositions of any embodiment and / or aspect disclosed herein, nucleic acids of any embodiment and / or aspect disclosed herein, viral vectors of any embodiment and / or aspect disclosed herein, lipid nanoparticles of any embodiment and / or aspect disclosed herein, cells of any embodiment and / or aspect disclosed herein, or pharmaceutical compositions of any embodiment and / or aspect disclosed herein in the manufacture of medicaments for treating, improving, or preventing diseases or conditions.

[0236] In several aspects, this disclosure provides a method for detecting and / or quantifying nucleic acids in a sample, the method comprising contacting the sample with a composition of any embodiment and / or aspect disclosed herein.

[0237] In one embodiment, the nucleic acid is a target and / or reporter nucleic acid. In another embodiment, the method includes detecting a reporter signal generated during cleavage by a nuclease. In yet another embodiment, the reporter signal is a fluorescent signal. In yet another embodiment, the nuclease has incidental cleavage activity.

[0238] In several aspects, this disclosure provides a variety of methods (e.g., using compositions of any of the embodiments and / or aspects disclosed herein). For example, compositions of any of the embodiments and / or aspects disclosed herein can be used to (i) modify (e.g., cleavage, such as nicking; methylation; etc.) target nucleic acids (DNA or RNA; single-stranded or double-stranded); (ii) regulate the transcription of target nucleic acids; (iii) label target nucleic acids; (iv) bind target nucleic acids (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify polypeptides (e.g., histones) associated with target nucleic acids; etc.

[0239] In embodiments, this disclosure provides a method for modifying a target nucleic acid. In embodiments, the method of this disclosure for modifying a target nucleic acid includes contacting the target nucleic acid with (a) a composition of this disclosure (e.g., an endonuclease and / or chimeric protein of any embodiment and / or aspect disclosed herein); and (b) one or more (e.g., two) RNAs of any embodiment and / or aspect disclosed herein. In embodiments, the method of this disclosure for modifying a target nucleic acid includes contacting the target nucleic acid with: a) a composition of this disclosure (e.g., an endonuclease and / or chimeric protein of any embodiment and / or aspect disclosed herein); and b) one or more (e.g., two) RNAs of any embodiment and / or aspect disclosed herein; and c) a donor nucleic acid (e.g., a donor template). In embodiments, the contacting step is performed in vitro in cells. In embodiments, the contacting step is performed in vivo in cells. In embodiments, the contacting step is performed in ex vivo cells.

[0240] It should be understood that, in implementations, while the binding method may simply result in the binding of the target nucleic acid, in other implementations the method may result in different end results (e.g., the method may result in modification of the target nucleic acid (e.g., cleavage / methylation, etc.); regulation of transcription from the target nucleic acid; regulation of translation of the target nucleic acid; genome editing; regulation of proteins associated with the target nucleic acid; isolation of the target nucleic acid; etc.).

[0241] For examples of suitable methods, see, for instance, Jinek et al., Science. Aug 17, 2012; 337(6096):816-21; Chylinski et al., RNA Biol. May 2013; 10(5):726-37; Ma et al., BiomedRes Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci US A. Sep 24, 2013; 110(39):15644-9; Jinek et al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. Sep 2013; 31(9):839-43; Qi et al., Cell. Feb 28, 2013; 152(5):1173-83; Wang et al., Cell. May 9, 2013; 153(4):910-8; Auer et al., Genome Res. October 31, 2013; Chen et al., Nucleic Acids Res. November 1, 2013; 41(20):e19; Cheng et al., Cell Res. October 2013; 23(10):1163-71; Cho et al., Genetics. November 2013; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. April 2013; 41(7):4336-43; Dickinson et al., NatMethods. October 2013; 10(10):1028-34; Ebina et al., Sci Rep. 2013;3:2510;Fujii et al., Nucleic Acids Res. 1 Nov 2013;41(20):e187;Hu et al., Cell Res. 1 Nov 2013;23(11):1322-5;Jiang et al., Nucleic Acids Res. 1 Nov 2013;41(20):e188;Larson et al., Nat Protoc. 1 Nov 2013;8(11):2180-96;Mali et al., Nat Methods. 1 Oct 2013;10(10):957-63;Nakayama et al., Genesis. 1 Dec 2013;51(12):835-43;Ran et al., NatProtoc. November 2013; 8(11):2281-308; Ran et al., Cell.September 12, 2013; 154(6):1380-9; Upadhyay et al., G3 (Bethesda). December 9, 2013; 3(12):2233-8; Walsh et al., Proc NatlAcad Sci US A. September 24, 2013; 110(39):15514-5; Xie et al., Mol Plant. October 9, 2013; Yang et al., Cell. September 12, 2013; 154(6):1370-9; and the following U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 2014 0186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140 273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 201403 35620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated in its entirety by reference.

[0242] In the implementation scheme, this disclosure provides (but is not limited to) methods for lysing target nucleic acids; methods for editing target nucleic acids; methods for regulating transcription from target nucleic acids; methods for isolating target nucleic acids; methods for binding target nucleic acids; methods for imaging target nucleic acids; methods for modifying target nucleic acids, etc.

[0243] In the embodiments, the term "contact a target nucleic acid" covers, for example, all methods for contacting a target nucleic acid. For instance, the polypeptides of the present invention (e.g., the endonucleases and / or chimeric proteins of the present invention) are provided to cells in the form of protein, RNA (encoding the polypeptides of the present invention), or DNA (encoding the polypeptides of the present invention); while the RNA of the present invention can be provided in the form of RNA or a nucleic acid encoding RNA. Therefore, when the method is performed, for example, in cells (e.g., inside cells in vitro, inside cells in vivo, inside cells in vitro), the method including contacting a target nucleic acid covers the introduction of any or all components in an active / final state (e.g., in the form of a protein polypeptide; in the form of a protein chimeric polypeptide; in the form of RNA of the present invention in the embodiments), and also covers the introduction of one or more nucleic acids encoding one or more components (e.g., nucleic acids containing a nucleotide sequence encoding the polypeptides of the present invention or the chimeric polypeptides of the present invention, nucleic acids containing a nucleotide sequence encoding a guide RNA, nucleic acids containing a nucleotide sequence encoding a donor template, etc.) into the cells. Because the methods described can also be performed outside of cells in vitro, methods that include contact with target nucleic acids (unless otherwise specified) cover contact outside of cells in vitro, inside cells in vitro, inside cells in vivo, and inside cells in vitro.

[0244] In embodiments, the method of this disclosure for modifying target nucleic acids includes introducing the present invention locus into target cells, for example, nucleic acids from cells containing the present invention locus (e.g., in embodiments, cells containing the present invention locus in their natural state (the state in which they occur in nature)), said nucleic acid comprising a nucleotide sequence encoding the present invention polypeptide and a nucleotide sequence of about 1 kb to 5 kb in length surrounding the present invention coding nucleotide sequence, wherein the target cells typically (in their natural state) do not contain the present invention locus. However, one or more spacer sequences (guide sequences encoding said coding crRNA) can be modified to target one or more target sequences of interest. Therefore, for example, in an embodiment, the method of this disclosure for modifying a target nucleic acid includes introducing the locus of the present invention into a target cell, for example, a nucleic acid obtained from a source cell (e.g., in an embodiment, a cell containing the locus of the present invention in its natural state (the state in which it occurs in nature)), wherein said nucleic acid has a length of 100 nucleotides (nt) to 5 kb (e.g., a length of 100 nt to 500 nt, 500 nt to 1 kb, 1 kb to 1.5 kb, 1.5 kb to 2 kb, 2 kb to 2.5 kb, 2.5 kb to 3 kb, 3 kb to 3.5 kb, 3.5 kb to 4 kb, or 4 kb to 5 kb) and contains a nucleotide sequence encoding the polypeptide of the present invention. As described above, in some such embodiments, one or more spacer sequences (guide sequences encoding the encoded crRNA) may be modified to target one or more target sequences of interest. In an embodiment, the method includes introducing into the target cell: i) the locus of the present invention; and ii) a donor DNA template. In one embodiment, the target nucleic acid is in an in vitro cell-free composition. In another embodiment, the target nucleic acid is present in target cells. In yet another embodiment, the target nucleic acid is present in target cells, wherein the target cells are eukaryotic cells. In yet another embodiment, the target nucleic acid is present in target cells, wherein the target cells are mammalian cells. In yet another embodiment, the target nucleic acid is present in target cells, wherein the target cells are plant cells.

[0245] Reverse splicing system and binding sequence

[0246] In an embodiment, this disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splicing donor and / or splicing acceptor.

[0247] Additionally, this disclosure provides a composition comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing.

[0248] In the implementation scheme, the trans-splicing system includes a splice donor and a splice acceptor, and the trans-splicing system is suitable for replacing internal exons in a gene.

[0249] In one embodiment, the trans-splicing system includes a repair template comprising a splice donor and / or a splice acceptor. In another embodiment, the trans-splicing system includes a repair template comprising a splice donor and a splice acceptor. In yet another embodiment, the trans-splicing system includes a repair template comprising a splice donor and a splice acceptor, and the trans-splicing system and / or repair template are suitable for replacing internal exons in a gene. In another embodiment, a method is provided for trans-splicing exons in a target nucleic acid (e.g., precursor mRNA) in a cell, the method comprising contacting the cell with a trans-splicing system disclosed herein, wherein the trans-splicing system includes a repair template comprising a splice donor and a splice acceptor. In yet another embodiment, this disclosure provides a method for trans-splicing exons in a target nucleic acid (e.g., precursor mRNA) in the cells of a subject in need, the method comprising applying an effective amount of the trans-splicing system disclosed herein, wherein the trans-splicing system includes a repair template comprising a splice donor and a splice acceptor.

[0250] In some embodiments, the trans-splicing system further includes a repair template lacking a splice donor and / or splice acceptor. In some embodiments, the trans-splicing system is suitable for splicing target nucleic acids containing splice donor or splice acceptor sites. In some embodiments, the trans-splicing system comprises RNA molecules, such as gRNA targeting splice acceptor sites. In some embodiments, the trans-splicing system comprises RNA molecules, such as gRNA targeting splice donor sites. In some embodiments, the trans-splicing system is regulated or modulated by a small molecule. In various embodiments, the small molecule is selected from abscisic acid (ABA), rapamycin (or rapamycin analogs), FK506, cyclosporine A, FK1012, gibberellin 3-AM, FKCsA, AP1903 / AP20187, and auxin. In some embodiments, the precursor mRNA is located at an intron-exon junction or an exon-intron junction.

[0251] In one embodiment, the trans-splicing system comprises a precursor trans-splicing (PTS) molecule, wherein the PTS molecule comprises: i) one or more guide RNAs (gRNAs) targeting precursor mRNAs; ii) an intron sequence having a splicing signal; and iii) a donor sequence encoding a gene product or a portion thereof encoding a gene of interest. In one embodiment, the one or more gRNAs are contained within the PTS and treated with an endonuclease. In another embodiment, the one or more gRNAs are contained within the PTS and are not treated with an endonuclease. In yet another embodiment, the one or more gRNAs are not contained within the PTS. In a further embodiment, the PTS is a repRNA.

[0252] In one embodiment, the trans-splicing system comprises a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing. In another embodiment, the trans-splicing system comprises a system for trans-splicing a target nucleic acid containing repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor. In another embodiment, the repRNA comprises one or more binding motifs that guide and / or hybridize the repRNA to a target nucleic acid molecule. In another embodiment, the repRNA further comprises a guide RNA (gRNA). In another embodiment, the gRNA hybridizes with the target nucleic acid molecule. In another embodiment, the gRNA guides the repRNA to the target nucleic acid molecule.

[0253] In one embodiment, the repRNA is operatively linked to one or more antisense sequences that bind to the target nucleic acid molecule. In another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In another embodiment, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is provided in a trans configuration to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule. In yet another embodiment, the repRNA is operatively linked to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide. In yet another embodiment, the repRNA is provided in a cis configuration to one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide.

[0254] In the implementation, the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding peptides, and is described herein as "grepRNA".

[0255] In this implementation, the grepRNA comprises repair RNA and gRNA. In this implementation, "grepRNA" may be used interchangeably with "PTS".

[0256] In one embodiment, the composition or system includes a splice acceptor. In another embodiment, the composition or system includes a splice donor.

[0257] In one embodiment, (a) at least one intron sequence, (b) a splice acceptor and / or splice donor sequence, and (c) at least one exon sequence are provided in cis or trans, or are suitably provided in cis or trans. In another embodiment, (a) at least one intron sequence, (b) a splice acceptor and / or splice donor sequence, and (c) at least one exon sequence are provided in trans, or are suitably provided in trans. In another embodiment, these elements are controlled by one or more promoters. In another embodiment, these elements are controlled by different promoters. In another embodiment, these elements are operatively linked but separated by cleavable sequences (e.g., self-cleaving ribozymes). In another embodiment, (i) multiple repRNA populations are controlled by different promoters, or (ii) a repRNA and another system member are controlled by different promoters.

[0258] In one embodiment, the one or more exons are or comprise one or more exons of the target nucleic acid molecule. In another embodiment, the one or more introns are or comprise one or more introns of the target nucleic acid molecule. In yet another embodiment, the one or more exons are or comprise one or more exons of the target nucleic acid molecule. In yet another embodiment, the one or more introns are or comprise one or more introns of the target nucleic acid molecule.

[0259] In one embodiment, the repRNA comprises one or more binding motifs that guide and / or hybridize the repRNA to a target nucleic acid molecule. In another embodiment, the one or more binding motifs bind and / or hybridize to the target nucleic acid molecule indirectly or directly. In yet another embodiment, the one or more binding motifs comprise an antisense sequence to the target nucleic acid molecule. In yet another embodiment, the one or more binding motifs hybridize to exons and / or introns or fragments thereof of the target nucleic acid. In yet another embodiment, the one or more binding motifs hybridize to fragments of exons of the target nucleic acid. In yet another embodiment, the one or more binding motifs hybridize to fragments of introns of the target nucleic acid.

[0260] In the embodiments, the one or more binding motifs comprise about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

[0261] In the embodiments, the one or more binding motifs are approximately 10-500 nucleotides, 15-500 nucleotides, 20-500 nucleotides, 30-500 nucleotides, 40-500 nucleotides, 50-500 nucleotides, or 60-500 nucleotides, or 70-500 nucleotides, or 80-500 nucleotides, or 90-500 nucleotides, or 100-500 nucleotides, or 100-400 nucleotides, or 100-300 nucleotides, or 100-200 nucleotides, or 200-400 nucleotides, or 200-300 nucleotides, or 300-400 nucleotides, or up to The number of nucleotides is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 150, 200, 250, 300, 400, or 500.

[0262] In an embodiment, the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any of SEQ ID NO: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0263] In one embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids. In another embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids, wherein the USH2A target nucleic acid is selected from exon 13 (SEQ ID NO: 2023) or intron 13 (SEQ ID NO: 2024). In another embodiment, the binding motif has molecular activity for binding USH2A target nucleic acids of SEQ ID NO: 2023 or SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein said fold change is measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2023 binding to any of SEQ ID NO: 804-2022 is greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the multiple change is measured based on the trans-splicing editing rate relative to the non-target rate.

[0264] In the implementation, the binding motif has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation, the binding motif has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5 or 2.5-3.0, said fold change being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2024 binding to any of SEQ ID NO: 804-2022 is greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the multiple change is measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation scheme, the molecular activity of the USH2A target nucleic acid of SEQ ID NO: 2024 binding to any of SEQ ID NO: 804-2022 is greater than a fold change of about 2.5-7.0, 3.0-7.0, 3.5-7.0, 4.0-7.0, 4.5-7.0, 5.0-7.0, 5.5-7.0, 6.0-7.0 or 6.5-7.0, or 2.5-6.5, 2.5-6.0, 2.5-5.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5 or 2.5-3.0, wherein the fold change is measured based on the trans-splicing editing rate relative to the non-target rate.

[0265] In one embodiment, the binding motif binds to the position of intron 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2024. In yet another embodiment, the binding motif binds to the USH2A target nucleic acid (SEQ ID NO: 2024). The positions of intron 13 of (2024), wherein the positions are selected from approximately positions 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000.In the implementation scheme, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to position 13 of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000.

[0266] In one embodiment, the binding motif binds to the position of intron 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2024. In yet another embodiment, the binding motif binds to the USH2A target nucleic acid (SEQ ID NO: 2024). The position of intron 13 of the target nucleic acid (2024), wherein the position relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, ... 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.In the implementation scheme, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to the USH2A target nucleic acid (SEQ ID NO: 804-2022). The position of intron 13 of (2024) relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450. 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.

[0267] In one embodiment, the binding motif binds to position 13 of exon 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2023. In yet another embodiment, the binding motif binds to position 13 of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80. In yet another embodiment, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to position 13 of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80.

[0268] In one embodiment, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid. In another embodiment, the USH2A target nucleic acid is SEQ ID NO: 2023. In yet another embodiment, the binding motif binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position relative to the splice donor site is selected from, but not limited to, approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80. In yet another embodiment, the binding motif is selected from any of SEQ ID NO: 804-2022 and binds to a position of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position relative to the splice donor site is selected from, but not limited to, approximately positions -10, -20, -30, -40, -50, -60, -70, or approximately -80.

[0269] In an embodiment, the binding motif is selected from SEQ ID NO: 804, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 6.0, 6.5, 7.0, or about 7.5, said fold change being measured based on the trans-splicing editing rate relative to the non-target rate. In one embodiment, the binding motif is SEQ ID NO: 804 and binds to a position in intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16, or relative to the splice donor site selected from approximately positions 5 to 20, approximately 8 to 16, or approximately 12 nucleotides from the splice donor site in intron 13 of USH2A. In another embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to a position in intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, approximately 5 to 20, 5 to 15, 5 to 10, or 8 to 16, or 12 to 16, or 14 to 16. In one embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from position 12. In another embodiment, the binding motif is selected from SEQ ID NO: 804 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, position 12.

[0270] In an embodiment, the binding motif is selected from SEQ ID NO: 804-818, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-818 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on the trans-splicing editing rate relative to the non-target rate. In the implementation, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from approximately positions 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130, or relative to the splice donor site selected from approximately positions 5 to 20, approximately 8 to 16, or approximately 12 nucleotides from the splice donor site in intron 13 of USH2A. In one embodiment, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, about 30 to 130, 35 to 130, 45 to 130, 50 to 130, 75 to 130, or 100 to 130. In another embodiment, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position is selected from positions 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123. In the implementation scheme, the binding motif is selected from SEQ ID NO: 804-818 and binds to the position of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024), wherein the position relative to the splice donor site is selected from, but not limited to, positions 38, 43, 95, 108, 110, 111, 112, 113, 115, 118, or 123.

[0271] In an embodiment, the binding motif is selected from SEQ ID NO: 804-883, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-883 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0272] In an embodiment, the binding motif is selected from SEQ ID NO: 804-1019, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-1019 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0273] In an embodiment, the binding motif is selected from SEQ ID NO: 804-1788, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion. In an embodiment, the binding motif of SEQ ID NO: 804-1788 has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a multiple of about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on a trans-splicing editing rate relative to the non-target rate.

[0274] In the implementation scheme, the binding motif is selected from SEQ ID NO: 804-1788 and combined to approximately positions 12, 38, 111, 115, 111, 123, 108, 110, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126, 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 10 9, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101, 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 122, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86, 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67, 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 8 4, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73, 94, 34, 840, 6, 430, 88, 27, 20, 38, 99, 11, 38, 129, 6, 30, 10, 135, 129, 69, 7, 2, 8, 102, 92, 36, 107, 32, 135, 22, 30, 28, 75, 6, 41, 34, 106, 146, 28, 22, 71, 22, 142, 32, 14, 73, 44, 850, 104, 126, 20, 36, 9, 27, 35, 138, 121, 170,92、36、890、104、12、870、14、38、34、34、69、72、11、72、16、67、136、143、91、38、1、21、137、83、61、20、36、104、29、8、30、5、84、30、106、78、16、148、9、25、144、87、36、74、44、9、34、14、32、4、6、35、34、76、44、77、73、2、6、2、138、30、850、62、5、137、12、89、39、40、23、79、141、74、30、540、130、14、15、20、23、12、77、146、10、8、2、13、42、35、28、730、24、143、62、4、41、24、37、92、10、840、29、8、45、138、660、38、11、20、39、90、90、107、31、135、37、61、22、2、142、37、4、6、40、87、38、140、38、59、6、29、139、15、9、145、640、15、131、13、141、2、26、18、106、4、32、62、21、14、100、820、60、8、3、33、12、22、7、59、76、47、9、30、59、730、860、26、143、230、540、32、27、54、136、36、17、10、22、36、250、590、155、150、12、105、81、36、8、850、34、35、210、52、840、22、21、18、91、4、820、52、29、28、660、31、63、85、880、18、18、78、890、640、155、34、93、4、81、890、78、28、24、14、22、20、24、70、210、24、147、153、10、34、139、18、19、0、16、470、140、14、220、630、71、26、22、23、15、28、105、93、85、14、420、146、31、8、39、37、35、16、10、530、6、78、21、22、47、60、28、8、77、0、19、78、85、20、660、21、4、45、4、12、5、24、154、31、390、470、4、25、8、7、6、152、140、220、55、1、25、29、4、3、27、14、6、190、45、24、13、46、18、12、46、32、151、410、7、20、350、3、40、620、39、0、880、30、22、155、24、0、157、32、1、8、142、14、440、160、16、64、530、870、147、160、240、17、33、8、2、68、39、33、141、137、76、2、19、400、76、860、58、19、23、8、860、360、7、8、51、820、14、12、1、190、6、26、450、16、150、52、1、47、18、24、27、210、18、57、36、33、650、4、80、70、32、26、16、46、150、29、17、250、600、35、56、60、48、23、37、19、0、154、0、180、27、26、4、830、25、58、149、410、7、152、49、0、160、380、600、16、56、590、0、28、440、58、2、630、53、158、31、2、650、16、240、84、4、57、330、20、16、70、9、62、360、26、26、17、153、50、14、350、2、26、68、52、200、154、18、32、153、27、3、0、53、460、560、600、1、159、16、120、18、0、230、28、730、360、54、1、30、72、105、158、390、390、540、48、880、370、32、17、4、49、36、18、8、500、50、26、2、23、610、25、6、200、145、4、3、450、55、16、80、570、6、460、670、17、152、76、560、16、71、20、74、156、14、7、74、0、220、550、18、15、530、18、350、21、450、400、720、2、66、0、0、630、500、33、14、4、670、6、68、2、370、151、80、50、54、180、7、0、16、560、74、158、22、900、0、1、420、180、31、60、70、270、830、190、280、320、83、78、49、50、51、14、8、420、58、75、650、148、46、70、156、44、900、151、7、720、159、570、370、380、8、720、12、41、270、6、260、200、156、920、52、12、280、33、580、70、0、157、460、72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25, or relative to the splice donor site, bound to approximately position 12, 38, 111, 115, 111, 123, 108, 1 10, 95, 111, 115, 113, 112, 118, 43, 122, 114, 108, 13, 109, 94, 124, 117, 88, 112, 109, 116, 127, 97, 79, 99, 134, 117, 126, 38, 97, 132, 12, 65, 95, 134, 126 28, 132, 123, 124, 110, 125, 116, 125, 96, 122, 127, 98, 90, 96, 86, 110, 103, 10, 12, 108, 109, 119, 118, 115, 118, 130, 87, 112, 67, 640, 129, 128, 10, 101 100, 80, 128, 89, 103, 38, 86, 64, 128, 123, 97, 94, 81, 66, 30, 430, 130, 131, 121, 113, 89, 119, 98, 65, 117, 107, 131, 42, 11, 5, 10, 20, 65, 148, 133, 32, 1 22, 124, 28, 80, 149, 103, 101, 28, 121, 42, 133, 96, 80, 82, 64, 99, 20, 38, 66, 82, 12, 98, 133, 113, 28, 24, 132, 24, 870, 37, 20, 139, 125, 10, 63, 119, 2, 86 102, 66, 5, 26, 40, 32, 95, 13, 10, 38, 20, 120, 34, 43, 22, 170, 79, 38, 24, 430, 93, 100, 43, 144, 82, 114, 34, 88, 6, 149, 102, 590, 39, 13, 69, 6, 22, 91, 67 101, 75, 4, 5, 230, 31, 2, 15, 440, 30, 63, 120, 116, 15, 10, 10, 36, 68, 61, 8, 84, 11, 24, 10, 74, 6, 170, 26, 20, 10, 41, 144, 136, 145, 64, 127, 34, 22, 36, 73,94、34、840、6、430、88、27、20、38、99、11、38、129、6、30、10、135、129、69、7、2、8、102、92、36、107、32、135、22、30、28、75、6、41、34、106、146、28、22、71、22、142、32、14、73、44、850、104、126、20、36、9、27、35、138、121、170、92、36、890、104、12、870、14、38、34、34、69、72、11、72、16、67、136、143、91、38、1、21、137、83、61、20、36、104、29、8、30、5、84、30、106、78、16、148、9、25、144、87、36、74、44、9、34、14、32、4、6、35、34、76、44、77、73、2、6、2、138、30、850、62、5、137、12、89、39、40、23、79、141、74、30、540、130、14、15、20、23、12、77、146、10、8、2、13、42、35、28、730、24、143、62、4、41、24、37、92、10、840、29、8、45、138、660、38、11、20、39、90、90、107、31、135、37、61、22、2、142、37、4、6、40、87、38、140、38、59、6、29、139、15、9、145、640、15、131、13、141、2、26、18、106、4、32、62、21、14、100、820、60、8、3、33、12、22、7、59、76、47、9、30、59、730、860、26、143、230、540、32、27、54、136、36、17、10、22、36、250、590、155、150、12、105、81、36、8、850、34、35、210、52、840、22、21、18、91、4、820、52、29、28、660、31、63、85、880、18、18、78、890、640、155、34、93、4、81、890、78、28、24、14、22、20、24、70、210、24、147、153、10、34、139、18、19、0、16、470、140、14、220、630、71、26、22、23、15、28、105、93、85、14、420、146、31、8、39、37、35、16、10、530、6、78、21、22、47、60、28、8、77、0、19、78、85、20、660、21、4、45、4、12、5、24、154、31、390、470、4、25、8、7、6、152、140、220、55、1、25、29、4、3、27、14、6、190、45、24、13、46、18、12、46、32、151、410、7、20、350、3、40、620、39、0、880、30、22、155、24、0、157、32、1、8、142、14、440、160、16、64、530、870、147、160、240、17、33、8、2、68、39、33、141、137、76、2、19、400、76、860、58、19、23、8、860、360、7、8、51、820、14、12、1、190、6、26、450、16、150、52、1、47、18、24、27、210、18、57、36、33、650、4、80、70、32、26、16、46、150、29、17、250、600、35、56、60、48、23、37、19、0、154、0、180、27、26、4、830、25、58、149、410、7、152、49、0、160、380、600、16、56、590、0、28、440、58、2、630、53、158、31、2、650、16、240、84、4、57、330、20、16、70、9、62、360、26、26、17、153、50、14、350、2、26、68、52、200、154、18、32、153、27、3、0、53、460、560、600、1、159、16、120、18、0、230、28、730、360、54、1、30、72、105、158、390、390、540、48、880、370、32、17、4、49、36、18、8、500、50、26、2、23、610、25、6、200、145、4、3、450、55、16、80、570、6、460、670、17、152、76、560、16、71、20、74、156、14、7、74、0、220、550、18、15、530、18、350、21、450、400、720、2、66、0、0、630、500、33、14、4、670、6、68、2、370、151、80、50、54、180、7、0、16、560、74、158、22、900、0、1、420, 180, 31, 60, 70, 270, 830, 190, 280, 320, 83, 78, 49, 50, 51, 14, 8, 420, 58, 75, 650, 148, 46, 70, 156, 44, 900, 151, 7, 720, 159, 570, 370, 380, 8, 720, 12, 41, 270, 6, 260, 200, 156, 920, 52, 12, 280, 33, 580, 70, 0, 157, 460, 72, 26, 22, 30, 54, 240, 260, 410, 380, 53, 48, 147, 30, 12, 620, 24, 62, 34, 5, 52, 550, 35, 830, 910, 34, 1, 9, 13, 910, 46, 68, 16, 48, 330, 320, 18, 24, 55, 0, 5, 18, 670, 270, 19, 400, 610, 57, 480, 510, 580, 570, 1, 0, 18, 44, 7, 470, 54, 3, 22, 66, 64, 610, or 25.

[0275] In the implementation scheme, the binding motif is any of those in Table 5.

[0276] In embodiments, the compositions or systems disclosed herein comprise one or more sequences that bind to and / or hybridize with RNA-binding peptides. In embodiments, the sequences binding to the RNA-binding peptides are assembled into a secondary structure suitable for interacting with the RNA-binding peptides. In embodiments, the secondary structure is or comprises a hairpin. In embodiments, the secondary structure is or comprises a stem, inner loop, multi-branched loop, or pseudoknot.

[0277] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0278] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0279] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0280] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0281] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0282] In an embodiment, the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

[0283] In embodiments, the compositions or systems disclosed herein comprise a nuclease, wherein the nuclease is a CRISPR-Cas enzyme.

[0284] In one implementation, the Cas is type I. In another implementation, the Cas is type IA, such as, but not limited to, Cas8a or Cas5. In another implementation, the Cas is type I. In another implementation, the Cas is type IB, such as, but not limited to, Cas8b. In another implementation, the Cas is type I. In another implementation, the Cas is type IC, such as, but not limited to, Cas8c. In another implementation, the Cas is type I. In another implementation, the Cas is type ID, such as, but not limited to, Cas10d. In another implementation, the Cas is type I. In another implementation, the Cas is type IE, such as, but not limited to, Cse1 or Cse2. In another implementation, the Cas is type I. In another implementation, the Cas is type IF, such as, but not limited to, Csy1, Csy2, or Csy3. In another implementation, the Cas is type I. In another implementation, the Cas is type IG, such as, but not limited to, GSU0054. In another implementation, the Cas is type I. In another implementation, the Cas type I is, but not limited to, Cas3.

[0285] In one embodiment, the Cas is type II. In another embodiment, the Cas is type II-A, such as, but not limited to, Csn2. In another embodiment, the Cas is type II. In another embodiment, the Cas is type II-B, such as, but not limited to, Cas4. In another embodiment, the Cas is type II. In another embodiment, the Cas is type II-C. In another embodiment, the Cas is type II. In another embodiment, the Cas type II is, but not limited to, Cas 9.

[0286] In one implementation, the Cas is type III. In another implementation, the Cas is type III-A, such as, but not limited to, Csm2. In another implementation, the Cas is type III. In another implementation, the Cas is type III-B, such as, but not limited to, Cmr5. In another implementation, the Cas is type III. In another implementation, the Cas is type III-C, such as, but not limited to, Cas10 or Csx11. In another implementation, the Cas is type III. In another implementation, the Cas is type III-D, such as, but not limited to, Csx10. In another implementation, the Cas is type III. In another implementation, the Cas is type III-E. In another implementation, the Cas is type III. In another implementation, the Cas is type III-F. In another implementation, the Cas is type III. In another implementation, the Cas type III is, but not limited to, Cas 10.

[0287] In one implementation, the Cas is type IV. In another implementation, the Cas is type IV-A. In yet another implementation, the Cas is type IV. In yet another implementation, the Cas is type IV-B. In yet another implementation, the Cas is type IV. In yet another implementation, the Cas is type IV-C.

[0288] In one implementation, the Cas is type V. In another implementation, the Cas is type VA, such as, but not limited to, Cas12a (Cpf1). In another implementation, the Cas is type V. In another implementation, the Cas is type VB, such as, but not limited to, Cas12b (C2c1). In another implementation, the Cas is type V. In another implementation, the Cas is type VC, such as, but not limited to, Cas12c (C2c3). In another implementation, the Cas is type V. In another implementation, the Cas is type VD, such as, but not limited to, Cas12d (CasY). In another implementation, the Cas is type V. In another implementation, the Cas is type VE, such as, but not limited to, Cas12e (CasX). In another implementation, the Cas is type V. In another implementation, the Cas is type VF, such as, but not limited to, Cas12f (Cas14 or C2c10). In another implementation, the Cas is type V. In another implementation, the Cas is type VG, such as, but not limited to, Cas12g. In another implementation, the Cas is type V. In another implementation, the Cas is type VH, such as, but not limited to, Cas12h. In one implementation, the Cas is type V. In another implementation, the Cas is type VI, such as, but not limited to, Cas12i. In another implementation, the Cas is type V. In another implementation, the Cas is type VK, such as, but not limited to, Cas12k (C2c5). In another implementation, the Cas is type V. In another implementation, the Cas is type VU, such as, but not limited to, C2c4, C2c8, or C2c9. In another implementation, the Cas is type V. In another implementation, the Cas V type is, but not limited to, Cas 12. In another implementation, the Cas is type VI.

[0289] In one embodiment, the Cas is type VI-A, such as, but not limited to, Cas13a (C2c2). In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-B, such as, but not limited to, Cas13b. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-C, such as, but not limited to, Cas13c. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-D, such as, but not limited to, Cas13d. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-X, such as, but not limited to, Cas13x.1. In another embodiment, the Cas is type VI. In another embodiment, the Cas is type VI-Y. In another embodiment, the Cas is type VI. In another embodiment, the Cas VI type is, but not limited to, Cas 13.

[0290] In the implementation scheme, the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10 or Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8, C2c9, Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d or Cas13x.1.

[0291] In embodiments, the compositions or systems disclosed herein comprise an RNA sequence that interacts with an active or catalytically inactivated endonuclease.

[0292] In one embodiment, the gRNA associates with one or more endonucleases, or is suitable for association with one or more endonucleases. In another embodiment, the endonuclease contains one or more mutations to reduce catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially inactivate the endonuclease catalytically relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations. In yet another embodiment, the one or more mutations increase catalytic activity relative to its unmutated form. In yet another embodiment, the endonuclease contains one or more mutations to substantially overactivate the endonuclease catalytically relative to its unmutated form. In the embodiments, the endonuclease comprises an amino acid sequence or a fragment or variant thereof of one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, and has at least about 70% identity with one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or has about 1 to about 20 amino acid modifications.

[0293] In embodiments, the compositions or systems disclosed herein comprise the endonucleases disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 106-130 or fragments or variants thereof), which are linked, associated with, and / or fused to an RNA-binding protein. In embodiments, the RNA-binding protein is a viral protein. In embodiments, the RNA-binding polypeptide is any RNA-binding polypeptide, optionally wherein the RNA-binding polypeptide is selected from MS2 capsid protein (MCP), PP7 capsid protein, PRR1, HgaII, Qβ capsid protein, IN protein, SLBP (stem-loop histone mRNA-binding protein), and M protein or variants thereof. In one embodiment, the RNA-binding protein is MS2. In another embodiment, the RNA-binding protein is the PP7 coat protein. In another embodiment, the RNA-binding protein is PRR1. In another embodiment, the RNA-binding protein is HgaII. In another embodiment, the RNA-binding protein is the Qβ coat protein. In another embodiment, the RNA-binding protein is an IN protein or an SLBP protein. In another embodiment, the RNA-binding protein is an M protein.

[0294] In some embodiments, the compositions or systems disclosed herein comprise an inactive Cas13K2F endonuclease fused to RBP. In some embodiments, the compositions or systems disclosed herein comprise an inactive Cas13K2F endonuclease fused to MS2 (“dCas13K2F-MS2”). In some embodiments, the compositions or systems disclosed herein comprise an inactive Cas13K2F endonuclease fused to PP7 (“dCas13K2F-PP7”).

[0295] In the implementation scheme, the trans-splicing nucleic acid template includes a splicing donor.

[0296] In the implementation scheme, the target nucleic acid includes a splice acceptor.

[0297] In one embodiment, this document discloses a method for targeted trans-splicing of precursor mRNA in cells, the method comprising contacting the cells with a system according to any of the embodiments disclosed herein.

[0298] In the implementation scheme, an RNA molecule containing a sequence complementary to one strand of the target nucleic acid molecule is a guide RNA (gRNA).

[0299] In the implementation scheme, the endonuclease is linked, associated with, and / or fused with the RNA-binding protein.

[0300] In the embodiments, the composition or system substantially prevents or eliminates cis-splicing of nucleic acids.

[0301] In the implementation plan, the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0302] In one embodiment, this document discloses a method for targeted trans-splicing of precursor mRNA in cells, the method comprising contacting the cells with a system according to any of the embodiments disclosed herein.

[0303] connector

[0304] In one embodiment, the endonuclease is linked, associated with, and / or fused with the RNA-binding protein. In another embodiment, the endonuclease is linked to the RNA-binding protein via a linker. In yet another embodiment, the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids. In yet another embodiment, the linker substantially comprises glycine and serine residues. In the implementation scheme, the connector is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS) n (n=1-4) (SEQ ID NO: 65) 、 (Gly)8 (SEQ ID NO: 66), (Gly)6 (SEQ ID NO: 67), (EAAAK) n (n=1-3) (SEQ ID NO: 68), A(EAAAK) nA (n = 2-5) (SEQ ID NO: 69), AEAAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75) or variants thereof, wherein the variants contain about 1, about 2, about 3, about 4, or about 5 mutations selected from substitutions or deletions.

[0305] Target nucleic acid

[0306] In one embodiment, the repRNA comprises a splicing donor. In another embodiment, the repRNA comprises a splicing acceptor.

[0307] In one embodiment, the repRNA contains exons of the target nucleic acid. In another embodiment, the repRNA contains introns of the target nucleic acid.

[0308] In one embodiment, the repRNA contains one or more non-natural introns. In another embodiment, the target nucleic acid is a precursor mRNA transcript molecule.

[0309] In one embodiment, the target nucleic acid is one or more Usher syndrome-related genes, fragments thereof, or precursor mRNA sequences thereof. In another embodiment, the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III. In another embodiment, the target nucleic acid is one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1, or precursor mRNA sequences thereof. In another embodiment, the target nucleic acid is one or more genes selected from the following: USH2A, GPR98, and DFNB31, or precursor mRNA sequences thereof. In another embodiment, the target nucleic acid is USH2A or its precursor mRNA sequence. In another embodiment, the target nucleic acid is exon 13 of the USH2A or its precursor mRNA sequence. In the implementation scheme, the target nucleic acid is a USH2A or its precursor mRNA sequence carrying the c.2299delG and / or c.2276G > T mutation.

[0310] In the implementation scheme, the target nucleic acid is referred to as the target sequence. In the implementation scheme, the target nucleic acid is or comprises a continuous nucleotide sequence present in the target RNA or target DNA. In the implementation scheme, a continuous nucleotide segment refers to a covalently linked and adjacent string of nucleotides. In the implementation scheme, the target nucleic acid has a length of or comprises at least about 10, 20, 30, 40, 50, 75, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500, 5,000, 7,500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 nucleotides. In the implementation scheme, the target nucleic acid has a length of or contains less than about 300,000, 250,000, 200,000, 100,000, 150,000, or 50,000 nucleotides. In the implementation scheme, the target nucleic acid has a length of or contains about 5-10, about 5-15, about 5-20, about 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50-150, about 50-200, about 50-250, about 50-300, about 100-200, about 100-300, about 200-300, about 1... 00-400, approximately 100-500, approximately 100-600, approximately 100-700, approximately 100-800, approximately 100-900, approximately 100-1000, approximately 200-400, approximately 200-500, approximately 200-600, approximately 200-700, approximately 200-800, approximately 200-900, approximately 200-1000, approximately 300-400, approximately 300-500, approximately 300-600, approximately 300-700, approximately 300-800 Approximately 300-900, Approximately 300-1000, Approximately 300-400, Approximately 300-500, Approximately 300-600, Approximately 300-700, Approximately 300-800, Approximately 300-900, Approximately 300-1000, Approximately 400-500, Approximately 400-600, Approximately 400-700, Approximately 400-800, Approximately 400-900, Approximately 400-1000, Approximately 500-600, Approximately 500-700, Approximately 500-800, Approximately 500 -900, approximately 500-1000, approximately 1,000-5,000, approximately 1,000-10,000, approximately 1,000-15,000, approximately 1,000-20,000, approximately 1,000-25,000, approximately 1,000-30,000, approximately 1,000-35,000, approximately 1,000-40,000, approximately 1,000-45,000, approximately 1,000-50,000, approximately 2,000-5,000, approximately 2,000-10,000, approximately 2,000-15,000, approximately 2,000-20,000, approximately 2,000-25,000, approximately 2,000-30,000, approximately 2,000-35,000, approximately 2,000-40,000, approximately 2,000-45,000, approximately 2,000-50,000, approximately 3,000-5,000, approximately 3,000-10,000, approximately 3,000-15,000, approximately 3,000-20,000, approximately 3,000-25,000, approximately 3,000-30,000, approximately 3,000-35,000, approximately 3,000-40,000, approximately 3,000-45,000, approximately 3,000-50,000, approximately 4,000-5,000, approximately 4,000-10,000, approximately 4,000-15,000, approximately 4,000-20,000, approximately 4,000-25,000, approximately 4,000-30,000, approximately 4,000-35,000, approximately 4,000-40,000 Approximately 4,000-45,000, approximately 4,000-50,000, approximately 5,000-10,000, approximately 5,000-15,000, approximately 5,000-20,000, approximately 5,000-25,000, approximately 5,000-30,000, approximately 5,000-35,000, approximately 5,000-40,000 Approximately 5,000-45,000, approximately 5,000-50,000, approximately 10,000-15,000, approximately 10,000-20,000, approximately 10,000-25,000, approximately 10,000-30,000, approximately 10,000-35,000, approximately 10,000-40,000, approximately 10,000 00-45,000, approximately 10,000-50,000, approximately 15,000-20,000, approximately 15,000-25,000, approximately 15,000-30,000, approximately 15,000-35,000, approximately 15,000-40,000, approximately 15,000-45,000, approximately 15,000-50 ,000, approximately 20,000-25,000, approximately 20,000-30,000, approximately 20,000-35,000, approximately 20,000-40,000, approximately 20,000-45,000, approximately 20,000-50,000, approximately 25,000-30,000, approximately 25,000-35,000 Approximately 25,000-40,000, approximately 25,000-45,000, approximately 25,000-50,000, approximately 30,000-35,000, approximately 30,000-40,000, approximately 30,000-45,000, approximately 30,000-50,000, approximately 35,000-40,000, approximately 35,000-45,000, approximately 35,000-50,000, approximately 40,000-45,000, or approximately 45,000-50,000 nucleotides.

[0311] In the implementation plan, the target nucleic acid is 10-50,000 nucleotides in length, for example, 10-45,000, 10-40,000, 10-35,000, 10-30,000, 10-20,000, 11-45,000, 11-40,000, 11-35,000, 11-30,000, 11-20,000, 12-45,000, 12-40,000, 12-35,000, 12-30,000, 12-25,000, 12-20,000, 13-45,000, 13-40,000, 13-35,000, 13-30,000, 13-25,000, 13-20,000. 0, 14-45,000, 14-40,000, 14-35,000, 14-30,000, 14-25,000, 14-20,000, 15-45,000, 15-40,000, 15-35,000, 15-30,000, 15-25,000, 15-20,000, 16- 45,000, 16-40,000, 16-35,000, 16-30,000, 16-25,000, 16-20,000, 17-45,000, 17-40,000, 17-35,000, 17-30,000, 17-25,000, 17-20,000, 18-45,000 0, 18-40,000, 18-35,000, 18-30,000, 18-25,000, 18-20,000, 19-45,000, 19-40,000, 19-35,000, 19-30,000, 19-25,000, 19-20,000, for example, length is 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 8 0, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,0 00, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or 50,000 nucleotides.

[0312] In one embodiment, the target nucleic acid is or contains ssRNA. In another embodiment, the target nucleic acid is or contains dsRNA. In yet another embodiment, the target nucleic acid is or contains ssDNA. In yet another embodiment, the target nucleic acid is or contains dsDNA. In yet another embodiment, the target nucleic acid is approximately 2 to approximately 6 nucleotides upstream of the PAM sequence.

[0313] In one implementation, the target nucleic acid is close to the exon. In another implementation, the target nucleic acid is upstream of the exon. In yet another implementation, the target nucleic acid is downstream of the exon. Finally, the target nucleic acid overlaps with the exon.

[0314] In one implementation, the target nucleic acid is close to the intron. In another implementation, the target nucleic acid is upstream of the intron. In yet another implementation, the target nucleic acid is downstream of the intron. Finally, the target nucleic acid overlaps with the intron.

[0315] Hybridization and / or combination

[0316] In an embodiment, this disclosure provides a composition comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; and (b) a splice donor and / or splice acceptor, wherein the repRNA is adapted for trans-splicing.

[0317] In an embodiment, this disclosure provides a system for trans-splicing a target nucleic acid comprising a repRNA, the repRNA comprising: (a) one or more exons and / or introns; and (b) a splicing donor and / or splicing acceptor.

[0318] In one embodiment, the hybridization is mediated by complete sequence complementarity with one strand of the target nucleic acid molecule. In another embodiment, the hybridization is mediated by partial sequence complementarity with one strand of the target nucleic acid molecule.

[0319] In the embodiments, the endonuclease comprises an amino acid sequence or a fragment or variant thereof of one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, and has at least about 70% identity with one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or has about 1 to about 20 amino acid modifications.

[0320] In the implementation scheme, the RNA-binding polypeptide and / or endonuclease bind to the target nucleic acid molecule.

[0321] In the embodiments, the composition or system substantially prevents or eliminates cis-splicing of nucleic acids.

[0322] In the implementation plan, the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

[0323] In an embodiment, the guide ribonucleic acid structure (i) comprises (a) a CRISPR RNA (crRNA) suitable for hybridization with a target nucleic acid molecule and / or (b) a trans-activating CRISPR RNA (tracrRNA) suitable for interaction with the endonuclease, or (ii) lacks (a) a crRNA suitable for hybridization with a target nucleic acid molecule and / or (b) a tracrRNA suitable for interaction with the endonuclease.

[0324] In the implementation scheme, the endonuclease repeat sequences used to prepare the guide RNA are selected from Table 3 below.

[0325] Table 3: Repetitive sequences of nucleases used to prepare guide RNA

[0326] In an embodiment, the guide RNA is or comprises a sequence of SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises a sequence of SEQ ID NO: 28, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 29 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 30 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In the implementation, the guide RNA is or contains a sequence of SEQ ID NO: 31 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it.In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 90 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 91 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 92 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In an embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 93 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In one embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 94 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In another embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 95 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it.In one embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 96 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it. In another embodiment, the guide RNA is or comprises the sequence of SEQ ID NO: 97 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it.

[0327] In the embodiments, the endonuclease is a polypeptide or fragment or variant of one of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or a nucleic acid polypeptide or fragment or variant of one of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130. In the embodiments, the endonuclease is guided to the target nucleic acid using guide RNA selected from SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97 or fragments or variants thereof, and / or the endonuclease is associated with guide RNA selected from SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97 or fragments or variants thereof.

[0328] In the implementation scheme, the endonuclease is SEQ ID NO: 1 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0329] In the implementation scheme, the endonuclease is SEQ ID NO: 2 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0330] In the implementation scheme, the endonuclease is SEQ ID NO: 3 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0331] In the implementation scheme, the endonuclease is SEQ ID NO: 4 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0332] In the implementation scheme, the endonuclease is SEQ ID NO: 80 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0333] In the implementation scheme, the endonuclease is SEQ ID NO: 81 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0334] In the implementation scheme, the endonuclease is SEQ ID NO: 82 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0335] In the implementation scheme, the endonuclease is SEQ ID NO: 83 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0336] In the implementation scheme, the endonuclease is SEQ ID NO: 84 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0337] In the implementation scheme, the endonuclease is SEQ ID NO: 85 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0338] In the implementation scheme, the endonuclease is SEQ ID NO: 86 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0339] In the implementation scheme, the endonuclease is SEQ ID NO: 87 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0340] In the implementation scheme, the endonuclease is SEQ ID NO: 88 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0341] In the implementation scheme, the endonuclease is SEQ ID NO: 89 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0342] In the implementation scheme, the endonuclease is SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 or SEQ ID NO: 97 or a fragment or variant thereof.

[0343] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 1 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28.

[0344] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 2 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 29.

[0345] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 3 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 30.

[0346] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 4 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 31.

[0347] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 80 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90.

[0348] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 81 or SEQ ID NO: 106-130, or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90.

[0349] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 82 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 90.

[0350] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 83 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 91.

[0351] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 84 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 92.

[0352] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 85 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 93 or SEQ ID NO: 94.

[0353] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 86 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28.

[0354] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 87 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 28.

[0355] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 88 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 95 or SEQ ID NO: 96.

[0356] In the implementation scheme, the endonuclease is any one of SEQ ID NO: 89 or SEQ ID NO: 106-130 or a fragment or variant thereof, and the guide RNA is SEQ ID NO: 97.

[0357] In one embodiment, the length of the RNA molecule is or contains at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In another embodiment, the length of the RNA molecule is or contains less than about 300, 250, 200, 100, 150, or 50 nucleotides. In the implementation scheme, the length of the RNA molecule is or comprises about 5-10, about 5-15, about 5-20, about 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50-150, about 50-200, about 50-250, about 50-300, about 100-200, about 100-300, or about 200-300 nucleotides.

[0358] In the implementation scheme, the length of the RNA molecule is 10-50 nucleotides, for example 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-3 0, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, for example, length It can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0359] In an embodiment, this disclosure provides a composition comprising a nucleic acid encoding a nuclease, the nuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and RNA containing a repetitive sequence having at least about 70% identity with one or more of SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97. In the embodiments, the composition has at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity with SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97, or has about 1 to about 20 nucleotide modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In one embodiment, the sequence contains at least one HEPN domain or a fragment or variant thereof. In another embodiment, the sequence contains at least two HEPN domains or fragments or variants thereof.

[0360] Nucleic acid modification

[0361] In embodiments, the nucleic acids disclosed herein have one or more modifications (e.g., base modifications, backbone modifications, etc.) to provide new or enhanced characteristics (e.g., improved stability) to the nucleic acids. Nucleosides are base-sugar combinations. The base moiety of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide is a nucleoside that also includes a phosphate ester group covalently linked to the sugar moiety of the nucleoside. For those nucleosides that include pentofuranose, the phosphate ester group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In the formation of oligonucleotides, the phosphate ester groups covalently link adjacent nucleosides to form a linear polymeric compound. Subsequently, the ends of such a linear polymeric compound may be further linked to form a cyclic compound; however, linear compounds are suitable. Additionally, linear compounds may have internal nucleotide base complementarity and thus can fold in a manner that produces fully or partially double-stranded compounds. Within oligonucleotides, the phosphate ester group is often referred to as the internucleotide backbone that forms the oligonucleotide. The normal bonds or backbone of RNA and DNA are 3' to 5' phosphodiester bonds.

[0362] In the implementation scheme, suitable nucleic acid modifications include, but are not limited to: nucleotides with 2'O-methyl modifications, nucleotides with 2'-fluorine modifications, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphate-thioester bonds, and 5' caps (e.g., 7-methylguanylic acid caps (m7G)). 2'-O-methyl modified nucleotides (also known as 2'-O-methylRNA) are naturally occurring RNA modifications found in tRNA and other small RNAs, appearing as post-transcriptional modifications. Oligonucleotides containing 2'-O-methylRNA can be synthesized directly. Without being bound by theory, this modification increases the Tm of the RNA:RNA duplex but only results in a minor change in RNA:DNA stability. It is stable against single-stranded ribonuclease attack and is generally about 5 to about 10 times less sensitive to DNase than DNA. 2'-fluorine modified nucleotides (e.g., 2'-fluorine bases) have fluorine-modified ribose, which increases binding affinity I and also confers a degree of relative nuclease resistance compared to native RNA. LNA bases modify the ribosome backbone by locking the bases in the C3'-inner position, which favors the A-type helical double-strand geometry of RNA. Unwilling to be bound by theory, this modification significantly increases Tm and also provides very strong nuclease resistance. Multiple LNA insertions can be placed anywhere in the oligonucleotide except the 3' end. Applications ranging from antisense oligonucleotides to hybridization probes to SNP detection and allele-specific PCR have been described. Due to the significant increase in Tm conferred by LNAs, they can also induce an increase in primer dimer formation and spontaneous clip formation. In embodiments, the number of LNAs incorporated into a single oligonucleotide is 10 bases or less. Phosphothioester (PS) bonds (e.g., phosphothioester linkages) replace non-bridging oxygen atoms in the phosphate backbone of nucleic acids (e.g., oligonucleotides) with sulfur atoms. Unwilling to be bound by theory, this modification makes internucleotide linkages resistant to nuclease degradation. Phosphothioester linkages can be introduced between the last 3-5 nucleotides at the 5' or 3' end of the oligonucleotide to inhibit exonuclease degradation. Including phosphate thioester bonds within oligonucleotides (e.g., throughout the entire oligonucleotide) can also help reduce endonuclease attack.

[0363] Linkage between the modified backbone and the modified nucleoside

[0364] In embodiments, the nucleic acids of the present invention optionally containing modifications include nucleic acids containing a modified backbone or non-natural nucleoside interlinking. In embodiments, nucleic acids having a modified backbone include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone.

[0365] In the embodiments, the oligonucleotide backbone containing a phosphorus atom includes, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), diaminophosphates, thiocarbonylaminophosphates, thiocarbonylalkylphosphonates, thiocarbonylalkyl phosphate triesters, selenophosphates and borophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these substances, and those with reverse polarity, wherein one or more nucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages. Suitable oligonucleotides with reverse polarity contain a single 3' to 3' linkage at the 3' terminal nucleotide linkage, i.e., it can be a single basic reverse nucleoside residue (nucleobase deletion or substitution with a hydroxyl group). It also includes various salts (such as potassium or sodium), mixed salts, and free acid forms.

[0366] In embodiments, the nucleic acids of the present invention comprise one or more thiophosphate and / or heteroatom nucleoside links, particularly -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (referred to as methylene (methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (wherein the native phosphodiester nucleotide link is represented as -OP(=O)(OH)-O-CH2-). MMI-type nucleoside links are disclosed in U.S. Patent No. 5,489,677, mentioned above, the disclosure of which is incorporated herein by reference in its entirety. Suitable amide nucleoside links are disclosed in U.S. Patent No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.

[0367] Suitable polynucleotide backbones, excluding phosphorus atoms, have backbones formed by short-chain alkyl or cycloalkyl nucleosides, mixed heteroatom and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatom or heterocyclic nucleosides. These include those with morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; riboacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

[0368] Simulation

[0369] In embodiments, the nucleic acids of the present invention are in the form of nucleic acid mimics or comprise nucleic acid mimics. In embodiments, when the term "mimic" is used for polynucleotides, the term is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced by non-furanose groups; the replacement of only the furanose ring is also referred to in the art as a sugar substitute. The heterocyclic base moiety is maintained or modified for hybridization with a suitable target nucleic acid. One such nucleic acid is a polynucleotide mimic that has shown excellent hybridization properties and is called a peptide nucleic acid (PNA). In PNA, the sugar backbone of the polynucleotide is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotide is retained and binds directly or indirectly to the nitrogen atom of the amide moiety of the backbone.

[0370] In the embodiments, the nucleic acid of the present invention is or comprises a morpholino-based analog (morpholino nucleic acid) having a heterocyclic base linked to a morpholino ring. It has been reported that numerous linking groups link the morpholino monomer unit in the morpholino nucleic acid. A class of linking groups has been selected to obtain nonionic oligomers. Nonionic morpholino-based oligomers are unlikely to have undesirable interactions with cellular proteins. Morpholino-based polynucleotides are nonionic analogs of oligonucleotides that are unlikely to form undesirable interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry (2002, 41(14), 4503-4510). Morpholin-based polynucleotides are disclosed in U.S. Patent No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. Various compounds within morpholino-based polynucleotides have been prepared, the compounds having various different linking groups connecting monomer subunits.

[0371] In the embodiments, the nucleic acid of the present invention is a mimic, said mimic being in the form of cyclohexenyl nucleic acid (CeNA) or containing cyclohexenyl nucleic acid (CeNA). The furanyl ring, normally present in DNA / RNA molecules, is replaced by a cyclohexenyl ring. CeNA DMT-protected phosphoramide monomers have been prepared and used for the synthesis of oligomers based on classical phosphoramide chemistry. Fully modified CeNA oligomers and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc.(The publication details of CeNA, 2000, 122, 8595-8602, are incorporated herein by reference in their entirety). Typically, the incorporation of CeNA monomers into the DNA strand increases the stability of the DNA / RNA hybrid. CeNA oligoadenylates form complexes with RNA and DNA complement that exhibit similar stability to the native complex. Studies demonstrating the incorporation of CeNA structures into native nucleic acid structures using NMR and circular dichroism continue to explore simple conformational modifications.

[0372] In the embodiments, the nucleic acid of the present invention comprises locked nucleic acid (LNA) wherein a 2'-hydroxyl group is attached to the 4' carbon atom of the sugar ring, thereby forming a 2'-C, 4'-C-oxymethylene bond, thereby forming a bicyclic sugar moiety. The bond may be methylene (-CH2-), the group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2 (Singh et al., Chem. Commun (The publication details of LNA are incorporated herein by reference in their entirety., 1998, 4, 455-456). LNA and LNA analogs exhibit very high double-stranded thermal stability (Tm = +3 to +10 °C) with complementary DNA and RNA, stability towards 3'-exonuclease degradation, and good solubility. Effective and non-toxic antisense oligonucleotides containing LNA have been described (e.g., Wahllestedt et al., 1998, 4, 455-456). Proc. Natl. Acad. Sci USA, 2000, 97, 5633-5638, the contents of which are incorporated herein by reference in their entirety.

[0373] Modified sugar portion

[0374] In embodiments, the nucleic acids of the present invention comprise one or more substituted sugar moieties. Suitable polynucleotides comprise sugar substituents selected from the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. O((CH2)) is particularly suitable. n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2, where n and m are 1 to approximately 10. Other suitable polynucleotides contain sugar substituents selected from the following: C1 to C2. 10Lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, aryl groups, O-alkaneyl groups or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkaneyl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta , 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety, namely alkoxyalkoxy. Another suitable modification includes 2'-dimethylaminoethoxy, namely O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), namely 2'-O-CH2-O-CH2-N(CH3)2.

[0375] Other suitable sugar substituents include methoxy (-O-CH3), aminopropoxy (--O CH2 CH2NH2), allyl (-CH2-CH=CH2), -O-allyyl (--O–CH2—CH=CH2), and fluorine (F). The 2'-sugar substituent can be at the arabinose (top) or ribose (bottom) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions on the oligomer, particularly at the 3' end nucleoside of the sugar or at the 3' position and 5' position of the 5' end nucleotide in 2'-5' linked oligonucleotides. The oligomer may also have sugar mimics that replace pentofuranose, such as the cyclobutyl moiety.

[0376] Base modification and substitution

[0377] In embodiments, the nucleic acids of the present invention comprise one or more nucleobases (often simply referred to in the art as “bases”) modified or substituted. In embodiments, as used herein, “unmodified” or “natural” nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine, and guanine 6-methyl derivatives and other alkyl derivatives, adenine and guanine 2-propyl derivatives and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil. Pyridine, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen (especially 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Other modified nucleobases include tricyclic pyrimidines, such as phenoxazincytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenthiazincytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazincytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido(4,5-b)indol-2-one), and pyridinoindolcytidine (H-pyridino(3',2':4,5)pyrrolo(2,3-d)pyrimido-2-one).

[0378] In embodiments, the heterocyclic base moiety may further include those in which the purine or pyrimidine base is replaced by another heterocycle, such as 7-deazo-adenine, 7-deazoguanosine, 2-aminopyridine, and 2-pyridone. Not wishing to be bound by theory, certain nucleobases can be used to increase the binding affinity of oligomers. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes and is a suitable base substitution, for example, when combined with 2'-O-methoxyethyl sugar modification.

[0379] Conjugate

[0380] In embodiments, the compositions of the present invention are chemically linked to one or more portions or conjugates to enhance the activity, cellular distribution, or cellular uptake of oligonucleotides. In embodiments, the portions or conjugates comprise conjugate groups covalently linked to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid esters, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or excretion of the nucleic acids of the present invention.

[0381] In embodiments, the conjugate may include a protein transduction domain or a PTD (also referred to as a CPP – cell-penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversal of lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. A PTD attached to another molecule (which may range from small polar molecules to large macromolecules and / or nanoparticles) facilitates membrane traversal, for example, from extracellular space to intracellular space or from cytosol to organelles (e.g., the nucleus). In embodiments, the PTD is covalently linked to the 3' end of a foreign polynucleotide. In embodiments, the PTD is covalently linked to the 5' end of a foreign polynucleotide.

[0382] Reagent test kit

[0383] In several aspects, this disclosure provides a kit comprising a container containing a composition or system of any embodiment and / or aspect of the present disclosure, a nucleic acid of any embodiment and / or aspect of the present disclosure, a viral vector of any embodiment and / or aspect of the present disclosure, a lipid nanoparticle of any embodiment and / or aspect of the present disclosure, a cell of any embodiment and / or aspect of the present disclosure, or a pharmaceutical composition of any embodiment and / or aspect of the present disclosure, and instructions for trans-splicing the nucleic acid.

[0384] In embodiments, this disclosure provides kits for performing the methods described herein. In embodiments, the kits comprise the compositions described herein, recombinant expression vectors, delivery systems, and / or pharmaceutical compositions described herein, optionally further comprising reagents for reconstitution and / or dilution.

[0385] General characteristics of the composition and system

[0386] In some embodiments, the composition further comprises a viral vector. In some embodiments, the viral vector is or comprises AAV. In some embodiments, the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9. In some embodiments, the composition further comprises a non-viral vector.

[0387] In embodiments, the compositions of the present invention are in the form of nanoparticles, or the delivery of the compositions of the present invention is achieved using nanoparticles, for example, any particles having a diameter of less than about 1000 nm. In embodiments, nanoparticles suitable for delivering the compositions of the present invention to target cells have a diameter of about 500 nm or less, for example, about 25 nm to about 35 nm, about 35 nm to about 50 nm, about 50 nm to about 75 nm, about 75 nm to about 100 nm, about 100 nm to about 150 nm, about 150 nm to about 200 nm, about 200 nm to about 300 nm, about 300 nm to about 400 nm, or about 400 nm to about 500 nm. In embodiments, nanoparticles suitable for delivering the compositions of the present invention to target cells have a diameter of about 25 nm to about 200 nm. In embodiments, nanoparticles suitable for delivery have a diameter of about 100 nm or less. In embodiments, nanoparticles suitable for delivery have a diameter of about 35 nm to about 60 nm.

[0388] In some embodiments, the composition further comprises lipid nanoparticles (LNPs), liposomes, lipid complexes, or polymer nanoparticles. In some embodiments, the LNPs comprise one or more of ionizable lipids, aminolipids, anionic lipids, neutral lipids, amphiphilic lipids, accessory lipids, structural lipids, PEG lipids, and lipids.

[0389] In an embodiment, a particle is provided, for example, a delivery particle comprising a lipid or lipid-like substance and a hydrophilic polymer (e.g., a cationic lipid and a hydrophilic polymer), wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-bistetradecanoyl-sn-glycerol-3-phosphocholine (DMPC) and / or the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or the particle further comprises cholesterol (e.g., particle from formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; formulation 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; formulation 3 = DOTAP 90, DMPC 0, PEG 5, cholesterol 5).

[0390] In embodiments, liposomes are used to deliver the compositions of this disclosure to target cells. Liposomes are spherical vesicle structures consisting of a single or multiple lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used in liposome formation. Although liposome formation is spontaneous when the lipid membrane is mixed with an aqueous solution, it can be accelerated by applying force in the form of shaking using a homogenizer, ultrasonic disruptor, or extrusion device. Several other additives can be added to liposomes to modify their structure and properties. For example, cholesterol or sphingomyelin can be added to liposome mixtures to help stabilize the liposome structure and prevent leakage of the internal cargo. Liposome formulations may primarily comprise natural phospholipids and lipids such as 1,2-distearate-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin choline, and monosialotetrahexosylganglioside.

[0391] In this embodiment, the composition is in the form of a lipid complex. Lipid complexes utilizing cationic lipids have been shown to be useful for gene transfer. Cationic lipids, due to their positive charge, naturally complex with negatively charged DNA. Furthermore, due to their charge, they interact with the cell membrane. Internalization of the lipid complex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also prevent the cell from degrading the DNA.

[0392] In the embodiments, the composition is in the form of a polymeric complex. Most polymeric complexes consist of cationic polymers, and their production is regulated by ionic interactions. A significant difference between the action methods of polymeric complexes and lipid complexes is that polymeric complexes cannot release their DNA load into the cytoplasm; for this to occur, co-transfection with an endosome lysin, such as an inactivated adenovirus (to dissolve endosomes generated during endocytosis), must take place. However, this is not always the case; polymers such as polyethyleneimine, like chitosan and trimethyl chitosan, have their own endosome disruption mechanisms.

[0393] In some embodiments, the composition is in the form of a dendritic polymer, i.e., a spherical, highly branched macromolecule, which can also be used for genetic modification of stem cells. The surface of the dendritic polymer particles can be functionalized to modify their properties. In particular, cationic dendritic polymers (e.g., dendritic polymers with a positive surface charge) may be constructed. When genetic material (such as DNA plasmids) is present, charge complementarity leads to temporary association of nucleic acids with the cationic dendritic polymer. The dendritic polymer-nucleic acid complex can be absorbed into the cell via endocytosis upon reaching its destination.

[0394] In several aspects, the composition or system component is a nucleic acid. In an embodiment, the RNA is or comprises mRNA or modified mRNA (mmRNA). In an embodiment, the DNA molecule is or comprises a vector or plasmid. In an embodiment, the nucleic acid comprises a codon-optimized sequence. In an embodiment, the nucleic acid comprises one or more modifications. In an embodiment, the modification is one or more of base modifications and backbone modifications.

[0395] In embodiments, sugar-based particles, such as GalNAc, can be used to deliver the compositions of this disclosure to target cells.

[0396] In several aspects, this disclosure provides a viral vector comprising nucleic acids of any of the embodiments and / or aspects disclosed herein. In several aspects, this disclosure provides an expression vector comprising nucleic acids of any of the embodiments and / or aspects disclosed herein.

[0397] In the implementation scheme, the expression vector is selected from viral expression vectors (e.g., viral vectors based on viruses such as vaccinia virus; poliovirus; adenovirus (see, for example, Li et al., Invest Opthalmol Vis Sci35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); adeno-associated virus (AAV) (see, for example, Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997; Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava, WO 93 / 09239; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, for example, Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc. In an embodiment, the recombinant expression vector of this disclosure is a recombinant adeno-associated virus (AAV) vector. In an embodiment, the recombinant expression vector of this disclosure is a recombinant lentiviral vector. In an embodiment, the recombinant expression vector of this disclosure is a recombinant retroviral vector.

[0398] In the implementation scheme, the viral vector is or contains AAV. In the implementation scheme, the AAV is or contains one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

[0399] In some embodiments, the composition further comprises a VLP, such as at least one structure with properties similar to a virus but not yet proven to be infectious. In some embodiments, the VLP is a non-replicating, non-infectious viral capsid containing a viral capsid but lacking all or part of the viral genome, particularly the replication components of the viral genome. In some embodiments, the VLP is composed of one or more viral proteins, such as, but not limited to, those referred to as capsids, shells, covers, surface, and structural proteins (e.g., VP1, VP2). In some embodiments, the VLP has a structure similar to a bacteriophage, is non-replicating and non-infectious, and lacks at least one or more genes encoding the replication mechanism of a bacteriophage, and also lacks one or more genes encoding one or more proteins responsible for viral attachment or entry into the host. In the embodiments, the VLP comprises a polypeptide that promotes or is suitable for VLP delivery, including but not limited to a retroviral gag polyprotein comprising a matrix polypeptide, a capsid polypeptide, and a nucleocapsid polypeptide (optionally having one or more heterologous protease cleavage sites among one or both of the following: e.g., TEV cleavage site, PreScission (a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage site, human rhinovirus 3C protease cleavage site, enterokinase cleavage site, Epstein-Barr virus protease cleavage site, cathepsin D cleavage site, and / or thrombin cleavage site); a matrix polypeptide and a capsid polypeptide; and a capsid polypeptide and a nucleocapsid polypeptide, such as a lentiviral gag polyprotein, such as bovine immunodeficiency virus gag polyprotein, murine leukemia virus (MLV). Gag proteins, simian immunodeficiency virus (MIV) gag polyproteins, feline MIV gag polyproteins, human MIV gag polyproteins, equine infectious anemia virus (EIV) gag polyproteins, and caprine arthritis encephalitis virus (CAPE) gag polyproteins, or gag polyproteins of alpha retroviruses, beta retroviruses, gamma retroviruses, delta retroviruses, ε retroviruses, or foamy viruses. In an embodiment, the polypeptide promoting or suitable for VLP delivery is co-delivered with a protease to promote the cleavage of the chimeric protein. In an embodiment, the cleavage of the chimeric protein occurs between the endonuclease and the polypeptide promoting or suitable for VLP delivery. In an embodiment, the protease is fused to the polypeptide promoting or suitable for VLP delivery.

[0400] In embodiments, depending on the host / vector system used, any of a variety of transcriptional and / or translational control elements may be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc. In embodiments, the nucleotide sequence encoding the RNA of the present invention is operatively linked to a control element, such as a transcriptional control element, like a promoter. In embodiments, the nucleotide sequence encoding the protein or fusion polypeptide of the present invention is operatively linked to a control element, such as a transcriptional control element, like a promoter. In embodiments, the transcriptional control element is a promoter. In embodiments, the promoter is a constitutively active promoter. In embodiments, the promoter is a tunable promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is a cell-type-specific promoter. In embodiments, the transcriptional control element (e.g., the promoter) is functional in the targeted cell type or the targeted cell population. For example, in an embodiment, the transcriptional control element is functional in eukaryotic cells (e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cells, bone marrow (BM) CD34(+) cells, etc.)). In an embodiment, eukaryotic promoters (promoters that are functional in eukaryotic cells) include EF1α; those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeat (LTR) sequences of retroviruses, and mouse metallothionein-I. The selection of appropriate vectors and promoters is entirely within the capabilities of those skilled in the art.

[0401] In one embodiment, the nucleotide sequence encoding the RNA of the present invention and / or the fusion polypeptide of the present invention is operatively linked to an inducible promoter. In another embodiment, the nucleotide sequence encoding the RNA of the present invention and / or the chimeric protein of the present invention is operatively linked to a constitutive promoter.

[0402] In the embodiments, the promoters are derived from viruses and may therefore be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. In the embodiments, the promoters are used to drive expression via any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter (e.g., is or contains SEQ ID NO: 76 or a variant thereof, such as the CMV immediate early promoter region (CMVIE)), the Rous sarcoma virus (RSV) promoter, the human U6 small nucleus promoter (U6) (Miyagishi et al., Nature Biotechnology 20, -97 - 500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 1 Sep 2003; 31(17)), the human H1 promoter (H1), etc.

[0403] In embodiments, the nucleotide sequence encoding the RNA of the present invention is operatively linked to (controlled by) a promoter operable in eukaryotic cells (e.g., the U6 promoter, enhanced U6 promoter, H1 promoter, etc.). Those skilled in the art will understand that when expressing RNA (e.g., guide RNA) from nucleic acids (e.g., expression vectors) using the U6 promoter (e.g., in eukaryotic cells) or another PolIII promoter, mutation of the RNA may be necessary if several consecutive Ts (encoding U in the RNA) are present. This is because a string of Ts in the DNA (e.g., about 5 Ts) can act as a terminator for polymerase III (PolIII). Therefore, to ensure transcription of the RNA in eukaryotic cells, it may be necessary to modify the sequence encoding said RNA to eliminate the T sequences. In embodiments, the nucleotide sequence encoding the protein of the present invention (e.g., the disclosed protein or chimeric protein) is operatively linked to a promoter operable in eukaryotic cells (e.g., the CMV promoter, EF1α promoter, estrogen receptor-regulated promoter, etc.).

[0404] In the implementation scheme, the inducible promoter includes, but is not limited to, one of the following: T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) regulated promoter, lactose-inducible promoter, heat shock promoter, tetracycline regulated promoter, steroid regulated promoter, metal regulated promoter, estrogen receptor regulated promoter, etc. Therefore, the inducible promoter can be regulated by molecules, including but not limited to doxycycline; estrogens and / or estrogen analogs; IPTG; etc. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., ahydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor protein (tetR), tetracycline operating sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinol / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and chelate metal ions) genes from yeast, mice, and humans), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-induced promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0405] In the implementation, the promoter is a spatially restricted promoter (e.g., a cell type-specific promoter, a tissue-specific promoter, etc.) such that, in a multicellular organism, the promoter is active (e.g., "ON") in a specific subset of cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter can be used, provided that the promoter is functional in the targeted host cell (e.g., a eukaryotic cell; a prokaryotic cell).

[0406] In this embodiment, the promoter is a reversible promoter. Suitable reversible promoters (including reversibly inducible promoters) are known in the art. Such reversible promoters can be isolated from and derived from many organisms, such as eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) for a second organism is well known in the art. Such reversible promoters and systems based on such reversible promoters but also containing additional control proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I). (alcA) gene promoters, promoters responsive to alcohol transactivator protein (AlcR), tetracycline-regulated promoters (e.g., promoter systems including Tet activator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulatory promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-induced promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthesis-induced promoters, etc.

[0407] In one embodiment, the vector contains a ribosome binding site for translation initiation and a transcription terminator. In another embodiment, the vector includes a suitable sequence for amplifying expression. In yet another embodiment, the vector includes a nucleotide sequence encoding a protein tag (e.g., a 6xHis tag, a hemagglutinin tag (e.g., GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO:77)), a fluorescent protein, etc.) which is fused to the protein of the present invention, for example, at the N-terminus or C-terminus or between the N-terminus and C-terminus, thereby producing a fused polypeptide of the present invention.

[0408] In the embodiments, methods for introducing nucleic acids (e.g., nucleic acids containing donor polynucleotide sequences, one or more nucleic acids encoding the proteins and / or RNAs of the present invention, etc.) into host cells are known in the art, and any convenient method is used to introduce nucleic acids (e.g., expression constructs) into cells. Suitable methods include, for example, viral infection, transfection, liposome transfection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0409] In the embodiments, the introduction of the recombinant expression vector into cells can occur in any culture medium and under any culture conditions that promote cell survival. In the embodiments, the introduction of the recombinant expression vector into target cells is performed in vivo or in vitro. In the embodiments, the introduction of the recombinant expression vector into target cells is performed in vitro.

[0410] In some embodiments, the proteins of the present invention (e.g., endonucleases, chimeric proteins) are provided in the form of nucleic acids. In some embodiments, the proteins of the present invention (e.g., endonucleases, chimeric proteins) are provided in the form of RNA. In some embodiments, the proteins of the present invention (e.g., endonucleases, chimeric proteins) are provided in the form of DNA. In some embodiments, the RNA is generated by direct chemical synthesis or can be transcribed in vitro from DNA (e.g., encoding the proteins of the present invention). Once synthesized, the RNA can be introduced into cells using any well-known technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0411] Methods for introducing nucleic acids into host cells are known in the art, and any convenient method can be used to introduce the compositions of the present invention into target cells (e.g., prokaryotic cells, eukaryotic cells, plant cells, animal cells, mammalian cells, human cells, etc.). Suitable methods include, for example, viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0412] In the implementation scheme, well-developed transfection techniques (see, for example, Angel and Yanik (2010) PloS ONE 5(7): e11756) and commercially available TRANSMESSENGER reagents from Qiagen, STEMFECT RNA transfection kits from Stemgent, and TRANSIT-mRNA transfection kits from Mirus Bio LLC can be used to deliver nucleic acids to cells. See also Beumer et al. (2008) PNAS 105(50):19821-19826.

[0413] In several aspects, this disclosure provides a lipid nanoparticle comprising nucleic acid of any embodiment and / or aspect disclosed herein.

[0414] In several aspects, this disclosure provides a cell comprising nucleic acids, viral vectors, or lipid nanoparticles of any of the embodiments and / or aspects disclosed herein.

[0415] In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a mammalian cell. In yet another embodiment, the cell is a human cell. In one embodiment, the cell is an immortalized cell. In yet another embodiment, the cell is harvested from a subject.

[0416] In the embodiments, the cells derived from the subject are derived from biological samples. In the embodiments, the biological samples include biopsies, tissues, or bodily fluids. In the embodiments, the biological samples include one or more of the following: tumor cells, cultured cells, stem cells, and differentiated cells. In the embodiments, a biological sample refers to a sample obtained from or derived from a source of interest (e.g., cells), as described herein. In the embodiments, the source of interest includes organisms, such as animals or humans. In the embodiments, the biological sample is biological tissue or fluid. Non-limiting examples of biological samples include bone marrow, blood, blood cells, ascites, (tissue or fine needle) biopsy samples, cell-containing body fluids, free-floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, swabs (e.g., skin swabs, vaginal swabs, oral swabs, and nasal swabs), lavage or irrigation fluids (such as catheter lavage or bronchoalveolar lavage), aspirates, scrapings, samples (e.g., bone marrow samples, tissue biopsy samples, and surgical samples), feces, other body fluids, secretions, and / or excretions, and the cells contained therein.

[0417] In embodiments, this disclosure provides a modified cell comprising a composition of the present disclosure. In embodiments, this disclosure provides a modified cell comprising a composition of the present disclosure, wherein the modified cell is a cell that normally does not contain a composition of the present disclosure. In embodiments, this disclosure provides a modified cell comprising nucleic acids (e.g., genetically modified cells), the nucleic acids comprising a nucleotide sequence encoding a composition of the present disclosure. In embodiments, a genetically modified cell is provided, the cell being genetically modified with mRNA comprising a nucleotide sequence encoding a composition of the present disclosure. In embodiments, a genetically modified cell is provided, the cell being genetically modified with a recombinant expression vector comprising a composition of the present disclosure.

[0418] In embodiments, the cells are primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. In embodiments, the cell used as the recipient of the composition of this disclosure is referred to as a "host cell" or "target cell". In embodiments, the host cell or target cell may be a recipient of the composition or system of this disclosure. The host cell or target cell may be a recipient of the RNP of this disclosure. The host cell or target cell may be a recipient of a single component of the k system of this disclosure.

[0419] Non-limiting examples of cells (target cells) include: eukaryotic cells, bacterial cells, archaea cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, lycophytes, hornworts, mosses, dicotyledons, monocotyledons, etc.), and algal cells (e.g., *Botrytis cinerea*, *Chlamydomonas reinhardtii*, marine algae). Cells can be derived from various organisms, including: *Sargassum fusiforme*, *Chlorella proteoglycans*, *Sargassum fusiforme*, *Pterygota*, seaweed (e.g., giant kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, cells derived from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells derived from mammals (e.g., ungulates (e.g., pigs, cattle, goats, sheep); rodents (e.g., rats, mice); non-human primates; humans; felines (e.g., cats); canines (e.g., dogs); etc.). In some embodiments, the cells are not derived from natural organisms (e.g., the cells may be synthetically produced cells; also known as artificial cells).

[0420] In some embodiments, the cell is an in vitro cell (e.g., an established cultured cell line). The cell may be an ex vivo cell (cultured cells from an individual). In some embodiments, the cell is an in vivo cell (e.g., cells within an individual). In some embodiments, the cell is an isolated cell. In some embodiments, the cell is a cell within an organism. In some embodiments, the cell is an organism. In some embodiments, the cell is a cell in a cell culture (e.g., an in vitro cell culture). In some embodiments, the cell is in a cell collection. In some embodiments, the cell is a prokaryotic cell or derived from a prokaryotic cell. Cell culture (e.g., an in vitro cell culture). In some embodiments, the cell is a cell in a cell collection. In some embodiments, the cell is a bacterial cell or may be derived from bacterial cells. In some embodiments, the cell is an archaea cell or derived from archaea cells. In some embodiments, the cell is a eukaryotic cell or derived from eukaryotic cells. In some embodiments, the cell is a plant cell or derived from plant cells. In some embodiments, the cell is an animal cell or derived from animal cells. In some embodiments, the cell is an invertebrate cell or derived from invertebrate cells. In some embodiments, the cell is a vertebrate cell or a cell derived from a vertebrate. In some embodiments, the cell is a mammalian cell or a cell derived from a mammal. In some embodiments, the cell is a rodent cell or a cell derived from a rodent. In some embodiments, the cell is a human cell or a cell derived from a human. In some embodiments, the cell is a microbial cell or a cell derived from a microbial cell. In some embodiments, the cell is a fungal cell or a cell derived from a fungal cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is an arthropod cell. In some embodiments, the cell is a protozoan cell.

[0421] In the implementation scheme, suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells); germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.); and somatic cells, such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, etc.

[0422] In the implementation plan, suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous transplanted and expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, hematopoietic stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, bone cells, fetal cells, undifferentiated cells, pluripotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0423] In some embodiments, the cells are immune cells, neurons, epithelial cells and endothelial cells, or stem cells. In some embodiments, the immune cells are T cells, B cells, monocytes, natural killer cells, dendritic cells, or macrophages. In some embodiments, the immune cells are cytotoxic T cells. In some embodiments, the immune cells are helper T cells. In some embodiments, the immune cells are regulatory T cells (Tregs).

[0424] In this embodiment, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells. Adult stem cells reside in differentiated tissues but retain the property of self-renewal and the ability to generate multiple cell types, which are typically the cell types in the tissue in which the stem cells reside. Many examples of somatic stem cells are known to those skilled in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and so on. Stem cells of interest include mammalian stem cells, where the term "mammal" means any animal classified as a mammal, including humans; non-human primates; livestock and farm animals; and zoo, laboratory, sport, or pet animals, such as dogs, horses, cats, cattle, mice, rats, rabbits, etc. In this embodiment, the stem cell is a human stem cell. In this embodiment, the stem cell is a rodent (e.g., mouse; rat) stem cell. In this embodiment, the stem cell is a non-human primate stem cell.

[0425] In the implementation scheme, the stem cells are hematopoietic stem cells (HSCs). HSCs are mesodermal cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized by CD34. + and CD3 -HSCs can regenerate erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewal cell division and differentiate into the same lineages observed in vivo. Therefore, HSCs can be induced to differentiate into one or more of erythroid, megakaryocyte, neutrophil, macrophage, and lymphoid cell types.

[0426] In this embodiment, the stem cells are neural stem cells (NSCs). Neural stem cells (NSCs) are capable of differentiating into neurons and glial cells (including oligodendrocytes and astrocytes). Neural stem cells are pluripotent stem cells capable of multiple divisions and, under certain conditions, can produce daughter cells as neural stem cells or neural progenitor cells as neuroblasts or glial cells, for example, cells dedicated to becoming one or more types of neurons and glial cells, respectively. Methods for obtaining NSCs are known in the art.

[0427] In this embodiment, the stem cells are mesenchymal stem cells (MSCs). MSCs originate from the embryonic mesoderm and are isolated from adult bone marrow, and can differentiate into muscle, bone, cartilage, fat, bone marrow matrix, and tendons. Methods for isolating MSCs are known in the art; and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0428] In the implementation plan, the cells are plant cells. For example, the cells can be cells of major agricultural plants, such as barley, beans (dried), rapeseed, corn, cotton (Pima cotton), cotton (upland cotton), flaxseed, hay (alfalfa), hay (non-alfalfa), oats, peanuts, rice, sorghum, soybeans, sugar beets, sugarcane, sunflower (oil), sunflower (non-oil), sweet potato, tobacco (burley tobacco), tobacco (flue-cured tobacco), tomato, wheat (durum wheat), wheat (spring wheat), wheat (winter wheat), etc. As another example, the cells are cells of vegetable crops, including but not limited to, alfalfa sprouts, aloe vera leaves, kudzu root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet leaves, beets, bitter melon, Chinese cabbage, broccoli, cauliflower (turnip), Brussels sprouts, cabbage, cabbage sprouts, cactus leaves (cactus fruit), zucchini, thistle, carrots, cauliflower, celery, chayote, artichokes, Chinese cabbage, Chinese celery, Chinese leeks, bok choy, chrysanthemum leaves (garland chrysanthemum), kale, corn stalks, sweet corn, cucumber, white radish, dandelion leaves, taro, pea shoots, and winter melon. Melon, eggplant, chicory, lettuce, fiddlehead fern, field celery, endive, mustard greens, kale, galangal (Siamese, Thai ginger), garlic, ginger root, burdock, tender leaves, Hanover salad greens, Mexican quinoa flowers (huauzontle), Jerusalem artichoke, jicama, kale tender leaves, turnip, white quinoa, lettuce (Baby lettuce), lettuce (Boston lettuce), lettuce (Boston red lettuce), lettuce (green leaves), lettuce (iceberg lettuce), lettuce (red lettuce), lettuce (green oak leaves), lettuce (red oak leaves), lettuce (processed lettuce), lettuce (red leaves), lettuce (Romagna lettuce), lettuce (red Roman lettuce), lettuce (Russian red mustard), linkok, white radish, long bean, lotus root, wild lettuce, agave leaves, yellow taro, mixed lettuce, mizuna, loofah (Smooth loofah), moo, hairy gourd, mushroom, mustard greens, yam, okra, water spinach, tender onion leaves, bottle gourd, ornamental corn, ornamental gourd, parsley, parsnip, pea, bell pepper, chili pepper, pumpkin, chicory, radish sprouts, radish, green purslane, green purslane, rhubarb, romaine lettuce, turnip, saltwort, loofah (horn-shaped / ridge-shaped loofah), spinach, pumpkin, straw bales, sugarcane, sweet potato, Chinese lettuce, tamarind, taro, taro leaves, taro sprouts, taro root, Mexican bean tree (Mexican acacia bean), red melon, physalis, tomato, cherry tomato, grape tomato, plum tomato, turmeric, tender turnip leaves, turnip, water chestnut, yampoo, yam, rapeseed, cassava, etc.

[0429] In this embodiment, the cell is an arthropod cell. For example, the cell may be a cell of a suborder, family, subfamily, tribe, subtribe, or species classified as follows: for example, a subphylum of chelicerates (…). Chelicerata ), multi-legged sub-gate ( Myriapodia Hexapods (Hexipodia) Arachnida ( Arachnida ), Insecta ( Insecta ), Lithoorders ( Archaeognatha ), Tachyonales ( Thysanura ), Paleoptera ( Palaeoptera ), Ephemerales ( Ephemeroptera ), Dragonflies ( Odonata Anisoptera ( Anisoptera ), Zygoptera ( Zygoptera Neopterygium ( Neoptera Exoptera ( Exopterygota ), Aeoloptera ( Plecoptera ), spinning foot mesh ( Embioptera ), Orthoptera ( Orthoptera ), Apoptera ( Zoraptera ), Dermoptera ( Dermaptera ), Dictyoptera ( Dictyoptera ), Blattales ( Notoptera ), Ceratopodidae ( Grylloblattidae Mantis ( ) Mantophasmatidae ), Phallothorax ( Phasmatodea ), Blattales ( Blattaria Isoptera ( ) Isoptera ), Mantises ( Mantodea ), Parapneuroptera , Rodents ( Psocoptera ), Thysanoptera ( Thysanoptera ), Phytophthora ( Phthiraptera ), Hemiptera ( Hemiptera ), endoptera ( Endopterygota ) or total metamorphosis ( Holometabola ), Hymenoptera ( Hymenoptera ), Coleoptera ( Coleoptera ), Torrentoptera ( Strepsiptera ), Ophiophyales ( Raphidioptera ), Euryptera ( Megaloptera ), Neuroptera ( Neuroptera ), Mecoptera ( Mecoptera ), Siberian ( Siphonaptera Diptera ( Diptera ), Trichoptera ( Trichoptera ) or Lepidoptera ( Lepidoptera ).

[0430] In this embodiment, the cell is an insect cell. For example, in this embodiment, the cell is a cell of a mosquito, grasshopper, hemiptera, fly, flea, bee, wasp, ant, lice, moth, or beetle.

[0431] In several aspects, this disclosure provides a pharmaceutical composition comprising a composition or system of any embodiment and / or aspect of the present disclosure, a nucleic acid of any embodiment and / or aspect of the present disclosure, a viral vector of any embodiment and / or aspect of the present disclosure, a lipid nanoparticle of any embodiment and / or aspect of the present disclosure, or a cell of any embodiment and / or aspect of the present disclosure, and a pharmaceutically acceptable carrier.

[0432] The compositions described herein may have a sufficiently basic functional group capable of reacting with inorganic or organic acids, or a carboxyl group capable of reacting with inorganic or organic bases, to form pharmaceutically acceptable salts. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, those formed from... Journal of Pharmaceutical Science , 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use Pharmaceutically acceptable salts are listed in PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated in their entirety by reference.

[0433] As non-limiting examples, pharmaceutically acceptable salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, dihydroxynaphthyl salts, phenylacetates, trifluoroacetates, acrylates, chlorobenzoates, dinitrobenzoates, and hydroxybenzene. Formate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyn-1,4-dicarboxylate, hexyn-1,4-dicarboxylate, decanoate, octanoate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, phthalate, terephthalate, propynate, propionate, phenylpropionate, sebate, octanoate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0434] The term "pharmaceutically acceptable salt" refers to a salt of the composition of the present invention having an acidic functional group (such as a carboxylic acid functional group) and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals (such as sodium, potassium, and lithium); hydroxides of alkaline earth metals (such as calcium and magnesium); hydroxides of other metals (such as aluminum and zinc); ammonia and organic amines, such as unsubstituted or hydroxylated mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tri-(2-OH-lower alkylamines), such as mono-, bis-, or tri-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tri-(hydroxymethyl)methylamine; N,N-di-lower alkyl-N-(hydroxy-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucosamine; and amino acids, such as arginine, lysine, etc.

[0435] In the implementation scheme, the compositions described herein are in a pharmaceutically acceptable salt form.

[0436] In various embodiments, the present invention relates to pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable carrier or excipient. Any pharmaceutical composition described herein may be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or excipient. Such compositions may optionally contain an appropriate amount of a pharmaceutically acceptable excipient to provide a form suitable for appropriate administration.

[0437] In various embodiments, the pharmaceutical excipients can be liquids, such as water and oils, including petroleum, animal, plant, or synthetically derived oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical excipients can be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. In embodiments, pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the agents described herein is administered intravenously. Aqueous saline solutions, dextran solutions, and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. Where necessary, any agent described herein may also contain small amounts of wetting agents, emulsifiers, or pH buffers. Other examples of suitable pharmaceutical excipients are described in [reference needed]. Remington's Pharmaceutical Sciences The references cited in 1447-1676 (edited by Alfonso R. Gennaro, 19th edition, 1995) are incorporated herein by reference.

[0438] This invention includes the pharmaceutical compositions (and / or additional therapeutic agents) described herein in various formulations. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of this invention described herein may be in the form of a solution, suspension, emulsion, drops, tablet, pill, aggregate, capsule, liquid-containing capsule, gelatin capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dried powder, or any other suitable form. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In one embodiment, the pharmaceutical composition is formulated as a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated as a gelatin capsule. In one embodiment, the pharmaceutical composition is formulated as a liquid.

[0439] If necessary, the pharmaceutical compositions (and / or additional agents) of the present invention may also include a solubilizer. Furthermore, the agents may be delivered using suitable media or delivery devices known in the art. The combination therapies outlined herein may be co-delivered in a single delivery media or delivery device.

[0440] Formulations comprising the pharmaceutical compositions (and / or other agents) of the present invention can be conveniently presented in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. Such methods typically involve the step of combining the therapeutic agent with a carrier constituting one or more excipients. Generally, the formulation is prepared by uniformly and tightly binding the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

[0441] In various embodiments, any pharmaceutical composition (and / or additional agents) described herein is formulated according to conventional procedures to a composition suitable for the administration mode described herein.

[0442] Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In one embodiment, administration is performed via parenteral injection. The mode of administration may be determined by the practitioner and depends in part on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

[0443] In embodiments, the compositions described herein are formulated into compositions suitable for oral administration according to conventional procedures. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as sweeteners, such as fructose, aspartame, or saccharin; flavoring agents, such as peppermint, wintergreen oil, or cherry; coloring agents; and preservatives to provide a pharmaceutically palatable formulation. Furthermore, in the case of tablets or pills, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over an extended period. Selective permeable membranes surrounding any of the compositions described herein, driven by osmotic activity, are also suitable for orally administered compositions. In these subsequent platforms, fluid from the environment surrounding the capsule is absorbed by the driving compound, which swells to displace the agent or agent composition through the pores. These delivery platforms provide a substantially zero-order delivery profile, rather than a cone-shaped profile of immediate release of the formulation. Delaying agents (such as glyceryl monostearate or glyceryl stearate) can also be useful. Oral compositions may include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In embodiments, the excipients are pharmaceutical grade. In addition to the active compound, the suspension may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum, and mixtures thereof.

[0444] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injections and infusions) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be prepared as sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. Components suitable for parenteral administration include sterile diluents such as water for injection, physiological saline solutions, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and agents for regulating tension, such as sodium chloride or dextrose.

[0445] For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cre...

Claims

1. A composition comprising a repair RNA (repRNA) sequence, said sequence comprising: (a) One or more exons and / or introns; (b) Splice donor and / or splice acceptor, The repRNA described therein is suitable for trans-splicing.

2. A system for trans-splicing a target nucleic acid comprising a repRNA, wherein the repRNA comprises: (a) One or more exons and / or introns; and (b) Splice donor and / or splice acceptor.

3. The composition of claim 1, wherein the one or more exons are or comprise one or more exons of a target nucleic acid molecule.

4. The composition of claim 1, wherein the one or more introns are or comprise one or more introns of a target nucleic acid molecule.

5. The system of claim 2, wherein the one or more exons are or comprise one or more exons of the target nucleic acid molecule.

6. The system of claim 2, wherein the one or more introns are or comprise one or more introns of the target nucleic acid molecule.

7. The composition or system of any one of claims 1-6, wherein the repRNA comprises one or more binding motifs that guide and / or hybridize the repRNA to a target nucleic acid molecule.

8. The composition or system of claim 7, wherein one or more binding motifs bind and / or hybridize to the target nucleic acid molecule indirectly or directly.

9. The composition or system of any one of claims 7-8, wherein the one or more binding motifs comprise a sequence antisense to the target nucleic acid molecule.

10. The composition or system of any one of claims 7-9, wherein the one or more binding motifs hybridize with exons and / or introns or fragments thereof of the target nucleic acid.

11. The composition or system of any one of claims 7-10, wherein the one or more binding motifs hybridize with a fragment of an exon of the target nucleic acid.

12. The composition or system of any one of claims 7-11, wherein the one or more binding motifs hybridize with fragments of introns of the target nucleic acid, optionally wherein the binding motif has molecular activity for binding the USH2A target nucleic acid of SEQ ID NO: 2024, said molecular activity being greater than a fold change of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said fold change being measured based on a trans-splicing editing rate relative to the non-target rate, optionally wherein the binding motif binds to the USH2A target nucleic acid (SEQ ID NO: 2024). The position of intron 13 of the target nucleic acid (2024), wherein the position relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460. 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000 nucleotides, optionally wherein the binding motif is selected from SEQ The nucleic acid contains any of the following: SEQ ID NO: 804-2022 and binds to position 13 of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024).The location relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 mm from the splice donor site in intron 13 of USH2A. 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.

13. The composition or system of any one of claims 7-12, wherein the one or more binding motifs comprise about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, or at least Approximately 9, at least approximately 10, at least approximately 11, at least approximately 12, at least approximately 13, at least approximately 14, at least approximately 15, at least approximately 16, at least approximately 17, at least approximately 18, at least approximately 19, at least approximately 20, at least approximately 21, at least approximately 22, at least approximately 23, at least approximately 24, at least approximately 25, at least approximately 26, at least approximately 27, at least approximately 28, at least approximately 29, at least approximately 30, at least approximately 50, at least approximately 75, at least approximately 100, at least approximately 150, at least approximately 200, at least approximately 250, or at least approximately 300 nucleotides, optionally including SEQ The polynucleotide sequence of any of SEQ ID NO: 804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion, or optionally containing the polynucleotide sequence of any of SEQ ID NO: 804, or SEQ ID NO: 804-818, or SEQ ID NO: 804-883, or SEQ ID NO: 804-1019, or SEQ ID NO: 804-1788.

14. The composition or system of any one of claims 7-13, wherein the one or more binding motifs are about 10-500 nucleotides, about 15-500 nucleotides, about 20-500 nucleotides, about 30-500 nucleotides, about 40-500 nucleotides, about 50-500 nucleotides, or about 60-500 nucleotides, or about 70-500 nucleotides, or about 80-500 nucleotides, or about 90-500 nucleotides, about 100-500 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, or about 300 nucleotides in the sequence that binds to and / or hybridizes with the RNA-binding polypeptide. -400 nucleotides, or at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 nucleotides.

15. The composition or system of any one of claims 1-14, wherein the composition or system comprises one or more sequences that bind to and / or hybridize with an RNA-binding polypeptide.

16. The composition or system of claim 15, wherein the sequence binding to the RNA-binding polypeptide is assembled into a secondary structure suitable for interacting with the RNA-binding polypeptide.

17. The composition or system of claim 16, wherein the secondary structure is or comprises a hair clip.

18. The composition or system of any one of claims 15-16, wherein the secondary structure is or comprises a stem, an inner ring, a multi-branched ring, or a pseudoknot.

19. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is a viral protein.

20. The composition or system of any one of claims 14-18, wherein the RNA-binding polypeptide is any RNA-binding polypeptide, optionally wherein the RNA-binding polypeptide is selected from MS2 capsid protein (MCP), PP7 capsid protein, PRR1, HgaII, Qβ capsid protein, IN protein, SLBP (stem-loop histone mRNA-binding protein), and M protein or variants thereof.

21. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is MS2.

22. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is the PP7 coat protein.

23. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is PRR1.

24. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is HgaII.

25. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is a Qβ coat protein.

26. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is the lN protein or the SLBP protein.

27. The composition or system of any one of claims 14-18, wherein the RNA-binding protein is the M protein.

28. The composition or system of any one of claims 7-27, wherein the one or more binding motifs comprise a recognition sequence for the formation of a ribonucleoprotein (RNP) complex.

29. The composition or system of any one of claims 7-28, wherein the one or more binding motifs comprise a sequence from small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA), optionally wherein the repRNA comprises a sequence from the snRNA or the snoRNA, optionally wherein the snRNA, snoRNA, protein forming the RNP or within the RNP, and / or nucleic acid encoding the protein forming the RNP or within the RNP comprises a modification or mutation that attenuates, weakens, reduces, diminishes, or eliminates RNP activity compared to the unmodified form, and / or results in attenuated RNA modification activity, wherein the RNP activity is optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation, compared to the unmodified form.

30. The composition or system of claim 29, wherein the snRNA, snoRNA, protein forming an RNP or within an RNP, and / or nucleic acid encoding the protein forming the RNP or within the RNP comprises at least one or more pseudouridine sites.

31. The composition or system of claim 29, wherein the snRNA, snoRNA, protein forming or within an RNP, and / or nucleic acid encoding the protein forming or within the RNP does not contain a pseudouridine site.

32. The composition or system of any one of claims 1-31, wherein the repRNA comprises at least one or more pseudouridine sites.

33. The composition or system of any one of claims 1-31, wherein the repRNA does not contain a pseudouridine site.

34. The composition or system of any one of claims 1-32, wherein the repRNA is modified to include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more pseudouridine sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of pseudouridine sites in the exon sequence.

35. The composition or system of claims 1-34, wherein the composition or system comprises repair RNA (repRNA) and / or a protein that forms or is within an RNP, the protein having the snRNA or snoRNA, or a nucleic acid encoding the protein that forms or is within the RNP, and / or a small RNA that induces the cleavage of RNA and / or CRISPR-Cas enzymes.

36. The composition or system of any one of claims 30-35, wherein the snRNA, snoRNA, protein forming an RNP or within an RNP, and / or nucleic acid encoding the protein forming the RNP or within the RNP comprises a modification or mutation that, compared to an unmodified form, increases, stimulates, or enhances RNP activity, or enhances RNA modification activity, wherein the RNP activity is optionally selected from cleavage, nucleic acid processing, pseudouridineization, and / or methylation.

37. The composition or system of claim 36, wherein the composition or system comprises, in cis or trans, repair RNA (repRNA) and / or a protein forming or within an RNP, the protein having the snRNA or snoRNA, or a nucleic acid encoding the protein forming or within the RNP, and / or a small RNA inducing the cleavage of RNA and / or CRISPR-Cas enzymes.

38. The composition or system of claim 37, wherein the cleavage is initiated by an RNP formed on the repRNA, or by an RNP formed in cis or trans.

39. The composition or system of any one of claims 1-38, wherein the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding polypeptides (e.g., "grepRNA").

40. The composition or system of any one of claims 1-39, wherein the composition or system comprises a shear acceptor; optionally, wherein the composition or system comprises a shear donor.

41. The composition or system of any one of claims 1-40, wherein the composition or system comprises (a) at least one intron sequence, (b) a splice acceptor and / or splice donor sequence, and (c) at least one exon sequence, said sequences being provided in cis or trans, or adapted to be provided in cis or trans.

42. The composition or system of any one of claims 1-40, wherein the (a) at least one intron sequence, (b) splice acceptor and / or splice donor sequence, and (c) at least one exon sequence are provided in trans form, or are suitable to be provided in trans form.

43. The composition or system of any one of claims 1-42, wherein the element is controlled by one or more promoters.

44. The composition or system of any one of claims 1-42, wherein the element is controlled by a different promoter.

45. The composition or system of any one of claims 1-44, wherein the elements are operatively connected but separated by cleavable sequences (e.g., self-cleaving ribozymes).

46. ​​The composition or system of any one of claims 1-45, wherein (i) a plurality of repRNA populations are controlled by different promoters, or (ii) the repRNA and another system member are controlled by different promoters.

47. The composition or system of any one of claims 29-46, wherein the snRNA comprises U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 or U11, or fragments or variants thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition or deletion.

48. The composition or system of any one of claims 29-47, wherein the snRNA or snoRNA is selected from any one of SEQ ID NO: 144-802, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion, optionally wherein SEQ ID NO: Any of 144-802 or a fragment or variant thereof forms an RNP complex, wherein the fragment or variant is optionally identical with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% and / or has about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, wherein the nucleic acid modifications are optionally selected from substitution, addition, or deletion.

49. The composition or system of any one of claims 1-48, wherein the repRNA further comprises guide RNA (gRNA), optionally wherein the composition or system comprises repair RNA (repRNA) and the small RNA comprises a modification or mutation that attenuates, weakens, reduces, diminishes, or eliminates activity compared to the unmodified form, or optionally wherein the composition or system comprises repair RNA (repRNA) and the small RNA comprises a modification or mutation that increases, stimulates, or enhances activity compared to the unmodified form, or optionally wherein the composition or system comprises repair RNA (repRNA) and a small RNA that induces RNA cleavage in cis or trans; optionally wherein the snRNA comprises M6A modification; optionally wherein the snRNA comprises M6A modification in cis or trans.

50. The composition or system of any one of claims 1-49, wherein the repRNA further comprises a ribozyme site; optionally, wherein the ribozyme site is a hairpin, hammerhead, hepatitis D virus (HDV), Varkud satellite (VS), glmS ribozyme site, twisted ribozyme site, or a variant thereof.

51. The composition or system of claim 50, wherein the ribozyme site is an HDV ribozyme site.

52. The composition or system of claim 50, wherein the ribozyme site is a twisted ribozyme site.

53. The composition or system of any one of claims 50-52, wherein the ribozyme site is upstream of one or more exons and / or introns of the repRNA.

54. The composition or system of any one of claims 50-52, wherein the ribozyme site is downstream of one or more exons and / or introns of the repRNA.

55. The composition or system of any one of claims 50-54, wherein the ribozyme site is upstream of the splice donor and / or splice acceptor of the repRNA.

56. The composition or system of any one of claims 50-55, wherein the ribozyme site is downstream of the splice donor and / or splice acceptor of the repRNA.

57. The composition or system of any one of claims 50-56, wherein the ribozyme cleaves the target.

58. The composition or system according to any one of claims 50-57, wherein the ribozyme is a trans-lysin.

59. The composition or system of any one of claims 1-58, wherein the repRNA comprises M6A modification; optionally, wherein the repRNA comprises M6A modification in cis or trans.

60. The composition or system of any one of claims 29-59, wherein the snRNA or snoRNA is modified to include at least one or more M6A sites.

61. The composition or system of any one of claims 29-60, wherein the snRNA or snoRNA is modified to include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the snRNA or snoRNA or (ii) the number of M6A sites in the exon sequence.

62. The composition or system of any one of claims 29-59, wherein the snRNA or snoRNA is modified to not contain an M6A site.

63. The composition or system of any one of claims 1-62, wherein the repRNA comprises at least one or more M6A sites.

64. The composition or system of any one of claims 1-63, wherein the repRNA is modified to include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more M6A sites than (i) the unmodified state of the repRNA or (ii) the number of M6A sites in the exon sequence.

65. The composition or system of any one of claims 1-62, wherein the repRNA does not contain an M6A site.

66. The composition or system of any one of claims 1-65, wherein the repRNA comprises a ribozyme site, the ribozyme site being cleaved at the 5' end of the repRNA.

67. The composition or system of any one of claims 1-65, wherein the repRNA comprises a ribozyme site, the ribozyme site being cleaved at the 3' end of the repRNA.

68. The composition or system of any one of claims 1-67, wherein the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 5' end of the repRNA.

69. The composition or system of any one of claims 1-68, wherein the repRNA comprises a ribozyme site that cleaves the snRNA or snoRNA at the 3' end of the repRNA.

70. The composition or system of any one of claims 1-69, wherein the composition or system further comprises at least one precursor rRNA stem-loop.

71. The composition or system of claim 70, wherein the at least one precursor rRNA stem loop has a 5' cap or a 3' polyA tail removed.

72. The composition or system of any one of claims 1-71, wherein the repRNA comprises at least one or more snRNA or snoRNA sequences.

73. The composition or system of claim 72, wherein the at least one or more snRNA or snoRNA sequences stabilize the repRNA.

74. The composition or system of any one of claims 1-73, wherein the repRNA comprises an artificial smU7 system; optionally, wherein the artificial smU7 system stabilizes the repRNA.

75. The composition or system of any one of claims 72-74, wherein the at least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 5' end of the snRNA or snoRNA.

76. The composition or system of claim 75, wherein the pseudoknot at the 5' end of the snRNA or snoRNA stabilizes the repRNA.

77. The composition or system of claim 75, wherein the at least one or more snRNA or snoRNA sequences comprise a pseudoknot at the 3' end of the snRNA or snoRNA.

78. The composition or system of claim 77, wherein the pseudoknot at the 3' end of the snRNA or snoRNA stabilizes the repRNA.

79. The composition or system of any one of claims 1-78, wherein multiple repRNAs are present and controlled by the same, different, or multiple promoters.

80. The composition or system of any one of claims 1-79, wherein the repRNA and one or more other components of the system of the present invention are controlled by the same or different promoters.

81. The composition or system of any one of claims 1-80, wherein the repRNA comprises an alternative promoter.

82. The composition or system of claim 81, wherein the repRNA comprises at least one or more alternative PolII promoters.

83. The composition or system of claim 82, wherein the one or more alternative Pol II promoters cap the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine), optionally wherein the one or more alternative Pol II promoters capping the 5' end of the repRNA with 7mG (7-methylguanosine) or TMG (tri-methylguanosine) stabilize the repRNA.

84. The composition or system of any one of claims 1-83, wherein the repRNA comprises at least one or more circularized 5' replacement splice donor (SD) repRNAs, optionally wherein the repRNA comprising at least one or more circularized 5' replacement splice donor (SD) repRNAs stabilizes the repRNA.

85. The composition or system of claim 84, wherein the repRNA comprising one or more circularized 5' substitution (SD) repRNAs has improved stability and resistance to exonucleases compared to the unmodified form.

86. The composition or system of claim 84 or 85, wherein the repRNA comprises at least one or more circularized 3' replacement splice acceptors (SA) repRNAs, optionally wherein the at least one or more circularized 3' replacement splice acceptors (SA) repRNAs stabilize the repRNA.

87. The composition or system of claim 86, wherein the repRNA comprising one or more circularized 3' substitution (SA) repRNAs has improved stability and resistance to exonucleases compared to the unmodified form.

88. The composition or system of any one of claims 1-87, wherein the repRNA comprises at least one or more circularized internal substitution (SD + SA) repRNAs, optionally wherein the at least one or more circularized internal substitution (SD + SA) repRNAs stabilize the repRNA.

89. The composition or system of claim 88, wherein the repRNA comprising one or more circularized internal substitution (SD + SA) repRNA has improved stability and resistance to exonucleases compared to the unmodified form.

90. The composition or system of any one of claims 49-89, wherein the gRNA hybridizes with the target nucleic acid molecule.

91. The composition or system of any one of claims 49-90, wherein the gRNA guides the repRNA to the target nucleic acid molecule.

92. The composition or system of any one of claims 49-91, wherein the guide RNA is or comprises a sequence of SEQ ID NO: 28-31 and / or SEQ ID NO: 90-97 or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with it.

93. The composition or system of any one of claims 49-92, wherein the guide RNA is or comprises about 10-300 nucleotides, about 15-300 nucleotides, about 20-300 nucleotides, about 30-300 nucleotides, about 40-300 nucleotides, about 50-300 nucleotides, about 60-300 nucleotides, about 100-300 nucleotides, about 200-300 nucleotides, about 100-200 nucleotides, about 50-200 nucleotides, or about 50-100 nucleotides, or at least about 5, at least about 6, at least about 7, or at least about 8 nucleotides. At least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides.

94. The composition or system of any one of claims 1-93, wherein the composition or system comprises a nuclease, wherein the nuclease is a CRISPR-Cas enzyme, optionally wherein the Cas is type I; optionally wherein the Cas is type IA, optionally wherein the Cas is Cas8a or Cas5; optionally wherein the Cas is type I; optionally wherein the Cas is type IB, optionally wherein the Cas is Cas8b; optionally wherein the Cas is type I; optionally wherein the Cas is type IC, optionally wherein the Cas is Cas8c; optionally wherein the Cas is type I; optionally wherein the Cas is type ID, optionally wherein the Cas is Cas10d; optionally wherein the Cas is type I; optionally wherein the Cas is type IE, optionally wherein the Cas is Cse1 or Cse2; optionally wherein the Cas is type I; optionally wherein the Cas is type IF, optionally wherein the Cas is Csy1, Csy2 or Csy3; optionally wherein the Cas is type I; optionally wherein the Cas is type IG, optionally wherein the Cas is GSU0054; optionally wherein the Cas is type I; optionally wherein the Cas is type I; optionally wherein the Cas is type I; Type I is, but not limited to, Cas3; or optionally, the Cas is Type II; optionally, the Cas is Type II-A, optionally, the Cas is Csn2; optionally, the Cas is Type II; optionally, the Cas is Type II-B, optionally, the Cas is Cas4; optionally, the Cas is Type II; optionally, the Cas is Type II-C; optionally, the Cas is Type II; optionally, the Cas Type II is, but not limited to, Cas. 9; or optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-A, optionally, wherein the Cas is Csm2; optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-B, optionally, wherein the Cas is Cmr5; optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-C, optionally, wherein the Cas is Cas10 or Csx11; optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-D, optionally, wherein the Cas is Csx10; optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-E; optionally, wherein the Cas is type III; optionally, wherein the Cas is type III-F; optionally, wherein the Cas is type III; optionally, wherein the Cas type III is, but not limited to, Cas10; or optionally, wherein the Cas is type IV; optionally, wherein the Cas is type IV-A; optionally, wherein the Cas is type IV;Optionally, the Cas is type IV-B; optionally, the Cas is type IV; optionally, the Cas is type IV-C; or optionally, the Cas is type V; optionally, the Cas is type VA, optionally, the Cas is Cas12a (Cpf1); optionally, the Cas is type V; optionally, the Cas is type VB, optionally, the Cas is Cas12b (C2c1); optionally, the Cas is type V; optionally, the Cas is type VC, optionally, the Cas is Cas12c (C2c3); optionally, the Cas is type V; optionally, the Cas is type VD, optionally, the Cas is Cas12d (CasY); optionally, the Cas is type V; optionally, the Cas is type VE, optionally, the Cas is Cas12e (CasX); optionally, the Cas is type V; optionally, the Cas is type VF, optionally, the Cas is Cas12f. (Cas14 or C2c10); optionally, the Cas is type V; optionally, the Cas is type VG, optionally, the Cas is Cas12g; optionally, the Cas is type V; optionally, the Cas is type VH, optionally, the Cas is Cas12h; optionally, the Cas is type V; optionally, the Cas is type VI, optionally, the Cas is Cas12i; optionally, the Cas is type V; optionally, the Cas is type VK, optionally, the Cas is Cas12k (C2c5); optionally, the Cas is type V; optionally, the Cas is type VU, optionally, the Cas is C2c4, C2c8, or C2c9; optionally, the Cas is type V; optionally, the Cas V type is, but not limited to, Cas 12; optionally, the Cas is type VI; or optionally, the Cas is type VI-A, optionally, the Cas is Cas13a (C2c2); Optionally, the Cas is type VI; Optionally, the Cas is type VI-B, Optionally, the Cas is Cas13b; Optionally, the Cas is type VI; Optionally, the Cas is type VI-C, Optionally, the Cas is Cas13c; Optionally, the Cas is type VI; Optionally, the Cas is type VI-D, Optionally, the Cas is Cas13d; Optionally, the Cas is type VI; Optionally, the Cas is type VI-X, Optionally, the Cas is Cas13x.1; Optionally, the Cas is type VI; Optionally, the Cas is type VI-Y; Optionally, the Cas is type VI;Optionally, the Cas VI type is, but not limited to, Cas13; or optionally, the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10 or Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8, C2c9, Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, or Cas13x.

1.

95. The composition or system of any one of claims 1-94, wherein the composition or system comprises an RNA sequence that interacts with an active or catalytically inactivated endonuclease.

96. The composition or system of any one of claims 49-95, wherein the gRNA is associated with one or more endonucleases, or is suitable for association with one or more endonucleases.

97. The composition or system of any one of claims 94-96, wherein the endonuclease comprises one or more mutations to reduce catalytic activity relative to the unmutated form.

98. The composition or system of any one of claims 94-97, wherein the endonuclease comprises one or more mutations to substantially catalytically inactivate the endonuclease relative to its unmutated form.

99. The composition or system of any one of claims 94-98, wherein the endonuclease comprises one or more mutations; optionally, wherein the one or more mutations increase catalytic activity relative to the unmutated form.

100. The composition or system of any one of claims 94-98, wherein the endonuclease comprises one or more mutations to make the endonuclease substantially over-catalyzed relative to the unmutated form.

101. The composition or system of any one of claims 94-100, wherein the endonuclease comprises an amino acid sequence of one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or a fragment or variant thereof, and has at least about 70% identity with one or more of SEQ ID NO: 1-4, SEQ ID NO: 80-89 and / or SEQ ID NO: 106-130, or has about 1 to about 20 amino acid modifications.

102. The composition or system of any one of claims 1-101, wherein the repRNA is operatively linked to one or more sequences antisense to the target nucleic acid molecule.

103. The composition or system of any one of claims 1-102, wherein the repRNA is provided in cis to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule.

104. The composition or system of any one of claims 1-103, wherein the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule.

105. The composition or system of any one of claims 1-104, wherein the repRNA is provided in trans form to one or more sequences that bind to and / or hybridize with the target nucleic acid molecule.

106. The composition or system of any one of claims 1-105, wherein the repRNA is operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding polypeptides.

107. The composition or system of any one of claims 1-106, wherein the repRNA is provided in cis to one or more sequences that bind to and / or hybridize with RNA-binding polypeptides.

108. The composition or system of any one of claims 1-105, wherein the repRNA is not operatively linked to one or more sequences that bind to and / or hybridize with RNA-binding polypeptides.

109. The composition or system of any one of claims 1-105, wherein the repRNA is provided in trans form to one or more sequences that bind to and / or hybridize with RNA-binding polypeptides.

110. The composition or system of any one of claims 1-109, wherein the repRNA is operatively linked to one or more recognition sequences for the formation of ribonucleoprotein (RNP) complexes.

111. The composition or system of any one of claims 1-109, wherein the repRNA is provided in cis to one or more recognition sequences for the formation of ribonucleoprotein (RNP) complexes.

112. The composition or system of any one of claims 1-109, wherein the repRNA is not operatively linked to one or more recognition sequences for the formation of ribonucleoprotein (RNP) complexes.

113. The composition or system of any one of claims 1-109, wherein the repRNA is provided in trans form to one or more recognition sequences for the formation of ribonucleoprotein (RNP) complexes.

114. The composition or system of any one of claims 1-113, wherein the repRNA is operatively linked to one or more gRNAs.

115. The composition or system of any one of claims 1-113, wherein the repRNA is provided in cis to one or more gRNAs.

116. The composition or system of any one of claims 1-113, wherein the repRNA is not operatively linked to one or more gRNAs.

117. The composition or system of any one of claims 1-113, wherein the repRNA is provided in a trans form to one or more gRNAs.

118. The composition or system of any one of claims 29-117, wherein the snRNA or snoRNA targets one or more exon splice enhancers (ESEs), one or more intron splice enhancers (ISEs), one or more exon splice silencers (ESSs) and / or one or more intron splice silencers (ISSs).

119. The composition or system of claims 29-118, wherein the snRNA or snoRNA comprises modifications to include at least one or more exon splicing enhancers (ESE), at least one or more intron splicing enhancers (ISE), at least one or more exon splicing silencers (ESS), and / or at least one or more intron splicing silencers (ISS).

120. The composition or system of any one of claims 1-119, further comprising a small RNA that induces RNA cleavage, said RNA optionally selected from one or more of the following: siRNA, small hairpin RNA (shRNA), U7 snRNA, U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and antisense oligonucleotides (ASO).

121. The composition or system of any one of claims 1-119, wherein the composition or system targets or is adapted to target one or more Usher syndrome-related genes, optionally wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II or Usher syndrome type III.

122. The composition or system of any one of claims 1-121, wherein the composition or system targets or is suitable for targeting one or more genes selected from the group consisting of CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1 or their precursor mRNA sequences.

123. The composition or system of claim 122, wherein the composition or system targets or is suitable for targeting one or more genes selected from: USH2A, GPR98 and DFNB31 or their precursor mRNA sequences.

124. The composition or system of any one of claims 1-123, wherein the composition or system targets or is adapted to target the USH2A or its precursor mRNA sequence.

125. The composition or system of any one of claims 1-124, wherein the composition or system targets or is adapted to target exon 13 of the USH2A or its precursor mRNA sequence.

126. The composition or system of any one of claims 1-125, wherein the composition or system targets or is suitable for replacing c. 2299delG and / or c. 2276G > T of the USH2A or its precursor mRNA sequence.

127. The composition or system of any one of claims 1-126, wherein the composition or system is suitable for correcting mutations or defects in one or more Usher syndrome-related genes.

128. The composition or system of any one of claims 121-127, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II or Usher syndrome type III.

129. The composition or system of any one of claims 1-128, wherein the composition or system is suitable for correcting mutations or defects in one or more genes selected from: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31 and CLRN1 or their precursor mRNA sequences.

130. The composition or system of claim 129, wherein the composition or system is suitable for correcting mutations or defects in one or more genes selected from USH2A, GPR98, and DFNB31 or their precursor mRNA sequences.

131. The composition or system of claim 130, wherein the composition or system is suitable for correcting mutations or defects in the USH2A or its precursor mRNA sequence.

132. The composition or system of any one of claims 128-131, wherein the composition or system is suitable for correcting mutations or defects in exon 13 of the USH2A or its precursor mRNA sequence.

133. The composition or system of any one of claims 128-132, wherein the composition or system is suitable for correcting c.2299delG and / or c.2276G > T of the USH2A or its precursor mRNA sequence.

134. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 131, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

135. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 132, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

136. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 133, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

137. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 134, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

138. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 135, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

139. The composition or system of any one of claims 1-133, wherein the repRNA comprises a polynucleotide sequence of SEQ ID NO: 136, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

140. The composition or system of any one of claims 94-139, wherein the endonuclease is linked, associated with, and / or fused with the RNA-binding protein.

141. The composition or system of any one of claims 94-140, wherein the endonuclease is linked to the RNA-binding protein via a adapter.

142. The composition or system of claim 141, wherein the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.

143. The composition or system of any one of claims 141-142, wherein the connector substantially comprises glycine and serine residues.

144. The composition or system according to any one of claims 141-143, wherein the connector is (GGS). n ,in n It can be 1, 2, 3, 4, or 5.

145. The composition or system according to any one of claims 141-144, wherein the connector is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS) n (n=1-4) (SEQ ID NO: 65) 、 (Gly)8 (SEQ ID NO: 66), (Gly)6 (SEQ ID NO: 67), (EAAAK) n (n=1-3) (SEQ ID NO: 68), A(EAAAK) n A (n = 2-5) (SEQ ID NO: 69), AEAAAAKEAAAKA (SEQ ID NO: 70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 71), PAPAP (SEQ ID NO: 72), KESGSVSSEQLAQFRSLD (SEQ ID NO: 73), EGKSSGSGSESSKST (SEQ ID NO: 74), and GSAGSAAGSGEF (SEQ ID NO: 75) or variants thereof, wherein the variants contain about 1, about 2, about 3, about 4, or about 5 mutations selected from substitution or deletion.

146. The composition or system of any one of claims 1-145, wherein the repRNA comprises the splicing donor.

147. The composition or system of any one of claims 1-145, wherein the repRNA comprises the splice acceptor.

148. The composition or system of any one of claims 1-147, wherein the repRNA comprises an exon of the target nucleic acid.

149. The composition or system of any one of claims 1-148, wherein the repRNA comprises an intron of the target nucleic acid.

150. The composition or system of any one of claims 1-149, wherein the repRNA comprises one or more non-natural introns.

151. The composition or system of any one of claims 2-150, wherein the target nucleic acid is a precursor mRNA transcript molecule.

152. The composition or system of any one of claims 1-151, wherein the repRNA comprises at least one intron spacer sequence, the intron spacer sequence comprising at least one ISE and ESS sequence.

153. The composition or system of claim 152, wherein the at least one intron spacer sequence comprising at least one ISE and ESS sequence increases the trans-splicing efficiency of the target RNA compared to the unmodified form.

154. The composition or system of claim 152 or 153, wherein the repRNA comprises at least one intron spacer sequence, the intron spacer sequence comprising at least one ISE and ESS sequence.

155. The composition or system of any one of claims 152-154, wherein the at least one intron spacer sequence comprising at least one ISE and ESS sequence reduces the trans-splicing efficiency of the target RNA compared to the unmodified form.

156. The composition or system of any one of claims 152-155, wherein the repRNA comprises at least one intron spacer sequence, the intron spacer sequence comprising at least one ISE and ESS sequence.

157. The composition or system of any one of claims 152-156, wherein the at least one intron spacer sequence comprising at least one ISE and ESS sequence increases the efficiency of off-target RNA trans-splicing compared to the unmodified form.

158. The composition or system of any one of claims 152-156, wherein the repRNA comprises at least one intron spacer sequence, the intron spacer sequence comprising at least one ISE and ESS sequence.

159. The composition or system of any one of claims 152-156 or 158, wherein the at least one intron spacer sequence comprising at least one ISE and ESS sequence reduces the efficiency of off-target RNA trans-splicing compared to the unmodified form.

160. The composition or system of any one of claims 1-159, wherein the repRNA comprises ESS, ESE, ISS and / or ISE sequences.

161. The composition or system of claim 160, wherein the repRNA targets one or more of ESS, ESE, ISS and / or ISE.

162. The composition or system of any one of claims 152-161, wherein the interaction, modulation and / or combination with one or more of the ESS, ESE, ISS and / or ISE reduces or eliminates the interaction, modulation and / or combination of one or more of the ESS, ESE, ISS and / or ISE with the target.

163. The composition or system of any one of claims 1-162, wherein the repRNA comprises an exon sequence having ESE and ESS sequences.

164. The composition or system of claim 163, wherein the exon sequences having ESE and ESS sequences increase or decrease trans-splicing efficiency against RNA targets compared to the unmodified form.

165. The composition or system of claim 163, wherein the repRNA comprises an exon sequence having ESE and ESS sequences.

166. The composition or system of claim 165, wherein the repRNA comprising exon sequences having ESE and ESS sequences increases or decreases the efficiency of trans-splicing against RNA off-target compared to the unmodified form.

167. The composition or system of any one of claims 1-166, wherein the repRNA comprises at least one or more G4 structures.

168. The composition or system of claim 167, wherein the repRNA comprises at least one or more G4 structures that isolate SD / SA motifs.

169. The composition or system of claim 167 or 168, wherein the G4 structure is unwound, for example by DHX36 or CNBP, and remains trapped in the unwound state in the presence of a complementary sequence (e.g., an endogenous target or exogenously delivered trigger RNA).

170. The composition or system of claims 167-169, wherein the G4 structure reduces off-target effects compared to the unmodified form.

171. The composition or system of any one of claims 1-170, wherein the repRNA comprises a modification, the modification comprising at least one or more scaffold sequences.

172. The composition or system of claim 171, wherein the at least one or more scaffold sequences mediate (e.g., recruit) the formation of condensate-like aggregates and / or improve the local concentration of repRNA compared to the unmodified form and other target proteins and / or RNA.

173. The composition or system of any one of claims 1-172, wherein the repRNA comprises a modification comprising at least one or more sequences for targeting the repRNA to a promoter of a target gene of interest, or to a proximal condensate that may contain the promoter.

174. The composition or system of claim 173, wherein the one or more sequences comprise enhancer RNA, snRNA, and / or snoRNA sequences.

175. The composition or system of any one of claims 1-174, wherein the repRNA comprises a modification, optionally wherein the modification improves the interaction and localization with the DNA sequence of the non-template strand of the target gene compared to the unmodified form.

176. The composition or system of claim 175, wherein the DNA sequence of the non-template strand of the target gene is a promoter, intron, exon, or enhancer.

177. The composition or system of claim 175 or 176, wherein the modification improves the interaction and localization with the DNA sequence of the non-template strand of the target gene by a protein-directed (e.g., transcription factor, dCas, ZNF or other RBP) or nucleotide-directed (e.g., R-loop) approach compared to the unmodified form.

178. The composition or system of any one of claims 1-177, wherein the repRNA comprises a modification, the modification comprising an additional RNA element, preferably wherein the additional RNA element improves subnuclear localization to nuclear spots compared to the unmodified form, thereby enhancing trans-splicing efficiency.

179. The composition or system of claim 178, wherein the additional RNA element comprises NEAT1 and / or MALAT1 or fragments thereof.

180. The composition or system of claim 179, wherein the additional RNA element comprises the nucleotide sequence of SEQ ID NO:803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

181. The composition or system of any one of claims 1-180, wherein the repRNA comprises modifications that enable it to target the transcription site of the target RNA.

182. The composition or system of any one of claims 1-181, wherein the repRNA comprises a modification comprising a 5' UTR or a 3' UTR modification; optionally wherein the modification comprising a 5' UTR or a 3' UTR modification alters intracellular or nuclear localization based on interaction with an endogenously or exogenously supplied molecule (e.g., the interaction of RNA G4 with transcription factors or other proteins localized to a specific cellular compartment).

183. The composition or system of any one of claims 1-182, wherein the repRNA comprises a modification in the 5' UTR of the repRNA.

184. The composition or system of claim 183, wherein the modification in the 5'UTR of the repRNA increases stability compared to the unmodified form.

185. The composition or system of claim 183, wherein the modification in the 5'UTR of the repRNA reduces stability compared to the unmodified form.

186. The composition or system of claims 183-185, wherein the modification in the 5' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form.

187. The composition or system of any one of claims 1-186, wherein the repRNA comprises a modification in the 3' UTR of the repRNA.

188. The composition or system of claim 187, wherein the modification in the 3'UTR of the repRNA increases stability compared to the unmodified form.

189. The composition or system of claim 188, wherein the modification in the 3'UTR of the repRNA reduces stability compared to the unmodified form.

190. The composition or system of claims 187-189, wherein the modification in the 3' UTR of the repRNA increases or decreases translation efficiency compared to the unmodified form.

191. The composition or system of any one of claims 1-190, wherein the repRNA comprises a modification comprising modifying the repRNA to include a G4 structure, the G4 structure mediating the recruitment of splice-related RBPs.

192. The composition or system of any one of claims 1-191, wherein the repRNA comprises a modification, the modification comprising at least one or more foothold switches in the repRNA.

193. The composition or system of claim 192, wherein the at least one or more foothold switches in the repRNA are conditionally activated or deactivated (e.g., SD / SA blocking, binding motif blocking, or RBP blocking) upon detection of endogenous or exogenous supply of target RNA.

194. The composition or system of any one of claims 1-193, wherein the repRNA comprises a modification, the modification comprising at least one or more complementary riboregulators (cis) in the repRNA.

195. The composition or system of claim 194, wherein at least one or more complementary riboregulators (cis) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

196. The composition or system of any one of claims 1-195, wherein the repRNA comprises a modification, the modification comprising at least one or more self-complementary riboregulators (cis) in the repRNA.

197. The composition or system of claim 196, wherein at least one or more self-complementary riboregulators (cis) in the repRNA block the splice acceptor (SA) site and reduce off-target trans-splicing.

198. The composition or system of any one of claims 1-197, wherein the repRNA comprises a modification, the modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA.

199. The composition or system of claim 198, wherein at least one or more self-complementary riboregulators (trans) in the repRNA block the splice donor (SD) site and reduce off-target trans splicing.

200. The composition or system of any one of claims 1-199, wherein the repRNA comprises a modification, the modification comprising at least one or more self-complementary riboregulators (trans) in the repRNA.

201. The composition or system of claim 200, wherein at least one or more self-complementary riboregulators (trans) in the repRNA block the splice acceptor (SA) site and reduce off-target trans splicing.

202. The composition or system of any one of claims 1-201, wherein the repRNA comprises a modification, the modification comprising at least one or more binding motifs.

203. The composition or system of claim 202, wherein the at least one or more binding motifs increase trans-splicing efficiency, target specificity, and target site blocking (SA, SD, ISS, ISE, ESE, and ESS) compared to the unmodified form.

204. The composition or system of any one of claims 1-203, wherein the repRNA comprises a modification that enables it to induce trans-splicing in response to a stimulus, compared to its unmodified form.

205. The composition or system of any one of claims 1-204, wherein the repRNA comprises a modification that causes it to shut down or reduce trans-splicing in response to a stimulus, compared to its unmodified form.

206. The composition or system of any one of claims 1-205, wherein the repRNA comprises a modification that enables small molecule-induced trans-splicing compared to its unmodified form.

207. The composition or system of any one of claims 1-205, wherein the repRNA comprises a modification that, compared to its unmodified form, inhibits small molecule-induced trans-splicing.

208. The composition or system of any one of claims 1-207, wherein the repRNA comprises a modification enabling it to perform light-induced trans-splicing.

209. The composition or system of any one of claims 1-208, wherein the repRNA comprises a modification, the modification comprising at least one or more motifs bound and regulated by a light-sensitive protein.

210. The composition or system of any one of claims 1-209, wherein the snRNA or snoRNA comprises a sequence in the 3' untranslated region (3'UTR), optionally wherein the sequence in the 3' untranslated region (3'UTR) increases trans-splicing efficiency compared to the unmodified form.

211. The composition or system of claim 210, wherein the sequence is derived from the MALAT1 gene.

212. The composition or system of claim 211, wherein the sequence is a nucleotide sequence of SEQ ID NO: 803, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

213. The composition or system of any one of claims 1-212, wherein the RNP is assembled on the repRNA and / or the target.

214. The composition or system of any one of claims 1-212, wherein the RNP is assembled on the repRNA.

215. The composition or system of any one of claims 1-212, wherein the RNP is assembled on the target.

216. The composition or system of any one of claims 1-215, wherein the RNP spatially blocks and inhibits cis-splicing.

217. The composition or system of any one of claims 1-216, wherein the repRNA comprises a minimal intron.

218. The composition or system of claim 217, wherein the minimum intron is less than about 50 nucleotides, less than about 60 nucleotides, less than about 70 nucleotides, less than about 80 nucleotides, less than about 90 nucleotides, less than about 100 nucleotides, less than about 110 nucleotides, less than about 120 nucleotides, less than about 130 nucleotides, less than about 140 nucleotides, or less than about 150 nucleotides, or about 50 to about 150 nucleotides, or about 50 to about 100 nucleotides, or about 50 to about 75 nucleotides, or about 75 to about 150 nucleotides, or about 100 to about 150 nucleotides, or about 120 to about 150 nucleotides.

219. The composition or system of any one of claims 2-218, wherein the target nucleic acid is one or more Usher syndrome-related genes or fragments thereof or precursor mRNA sequences thereof.

220. The composition or system of claim 219, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II or Usher syndrome type III.

221. The composition or system of claim 220, wherein the target nucleic acid is one or more genes selected from the following: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31 and CLRN1 or their precursor mRNA sequences.

222. The composition or system of claim 221, wherein the target nucleic acid is one or more genes selected from USH2A, GPR98 and DFNB31 or their precursor mRNA sequences.

223. The composition or system of any one of claims 219-222, wherein the target nucleic acid is USH2A or its precursor mRNA sequence.

224. The composition or system of any one of claims 219-223, wherein the target nucleic acid is exon 13 of USH2A or its precursor mRNA sequence.

225. The composition or system of any one of claims 219-224, wherein the target nucleic acid is a USH2A or its precursor mRNA sequence carrying the c.2299delG and / or c.2276G > T mutation.

226. The composition or system of any one of claims 8-225, wherein the hybridization is mediated by complete sequence complementarity with one strand of the target nucleic acid molecule.

227. The composition or system of any one of claims 8-225, wherein the hybridization is mediated by sequence complementarity with a portion of one strand of the target nucleic acid molecule.

228. The composition or system of any one of claims 1-227, wherein the repRNA mediates the generation of the trans-splicing of the corrected and / or wild-type USH2A nucleic acid gene transcript.

229. The composition or system of any one of claims 1-228, wherein the composition or system substantially prevents or eliminates cis-splicing of nucleic acids.

230. The composition or system of any one of claims 2-229, wherein the trans-splicing system targets at least one of intron 12, exon 13, and intron 13 of the USH2A nucleic acid sequence.

231. The composition or system of any one of claims 1-230, wherein the composition or system further comprises a viral vector or a non-viral vector.

232. The composition or system of claim 231, wherein the viral vector is or comprises AAV, and optionally wherein the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

233. The composition or system according to any one of claims 1-232, wherein the composition or system further comprises lipid nanoparticles (LNP), liposomes, lipid complexes or polymer nanoparticles.

234. The composition or system of claim 233, wherein the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphiphilic lipids, accessory lipids, structural lipids, PEG lipids, and lipids.

235. The composition or system according to any one of claims 1-234, wherein the component of the composition or system is a nucleic acid.

236. The composition or system of any one of claims 1-235, wherein the components of the composition or system comprise DNA molecules or RNA molecules.

237. The composition or system of claim 236, wherein the RNA is or comprises mRNA or modified mRNA (mmRNA).

238. The composition or system of claim 236, wherein the DNA molecule is or comprises a vector or plasmid.

239. The composition or system of any one of claims 235-237, wherein the nucleic acid comprises a codon-optimized sequence.

240. The composition or system of any one of claims 235-239, wherein the nucleic acid comprises one or more modifications.

241. The composition or system of claim 240, wherein the modification is one or more of base modification and skeleton modification.

242. A cell comprising a nucleic acid as described in any one of claims 235-241, a viral vector as described in claims 231-232, or lipid nanoparticles as described in claims 233-234.

243. The cell of claim 242, wherein the cell is a eukaryotic cell.

244. The cell of claim 242, wherein the cell is a mammalian cell.

245. The cell of claim 242, wherein the cell is a human cell.

246. The cell of claim 242, wherein the cell is an immortalized cell.

247. The cell of claim 242, wherein the cell is harvested from the subject.

248. A pharmaceutical composition comprising a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, lipid nanoparticles as described in claims 233-234, or cells as described in any one of claims 242-247, and a pharmaceutically acceptable carrier.

249. A kit comprising a container, the kit comprising a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, lipid nanoparticles as described in claims 233-234, or cells as described in any one of claims 242-247, and instructions for trans-splicing the nucleic acid.

250. A method for targeted trans-splicing of USH2A precursor mRNA in cells, the method comprising contacting the cells with a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, a lipid nanoparticle as described in claims 233-234, or a cell as described in any one of claims 242-247.

251. A method for targeted trans-splicing of precursor mRNA in cells, the method comprising contacting the cells with a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, a lipid nanoparticle as described in claims 233-234, or a cell as described in any one of claims 242-247.

252. The method of claim 250 or 251, wherein the method further comprises slowing down, reducing or eliminating transcription and / or stimulation of the target gene, enhancing or increasing Pol II arrest / release.

253. The method of any one of claims 250-252, wherein, compared to the unmodified form, the method increases trans-splicing by forcing the formation of a large G4 or similar RNA-RNA motif downstream of the binding motif of the repRNA.

254. The method of any one of claims 250-253, wherein the method further comprises modifying and converting endogenous RNA (e.g., but not limited to precursor mRNA, mRNA, lncRNA) into repRNA for trans splicing.

255. A method of treating a patient suffering from a disease associated with a mutation in the USH2A gene, the method comprising administering a therapeutically effective amount of a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, lipid nanoparticles as described in claims 233-234, or cells as described in any one of claims 242-247.

256. A method for treating, improving, or preventing a disease associated with a USH2A gene mutation, the method comprising: (a) Contacting cells with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticles of any one of claims 233-234, or the cells of any one of claims 242-247, and (b) Administer an effective amount of the cells to the subject.

257. A method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising administering to the subject an effective amount of a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, a lipid nanoparticle as described in claims 233-234, or a cell as described in any one of claims 242-247.

258. A method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising: (a) Contacting cells with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticles of any one of claims 233-234, or the cells of any one of claims 242-247, and (b) Administer an effective amount of the cells to the subject.

259. The method of any one of claims 250-258, wherein the cells are derived from the subject.

260. The method of any one of claims 250-259, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II or Usher syndrome type III.

261. The method of claim 260, wherein the Usher syndrome is Usher syndrome type I.

262. The method of claim 260, wherein the Usher syndrome is Usher syndrome type II.

263. The method of claim 260, wherein the Usher syndrome is Usher syndrome type III.

264. The method of any one of claims 250-263, wherein the method targets one or more Usher syndrome-related genes.

265. The method of claim 264, wherein the method targets one or more genes selected from the group consisting of: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

266. The method of claim 265, wherein the method targets one or more of USH2A, GPR98, and DFNB31.

267. The method of claim 265 or 266, wherein the method targets USH2A.

268. The method of any one of claims 250-267, wherein the method corrects mutations or defects in one or more Usher syndrome-related genes.

269. The method of claims 250-268, wherein the method corrects mutations or defects in one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

270. The method of claim 269, wherein the method corrects mutations or defects in one or more of USH2A, GPR98, and DFNB31.

271. The method of claim 270, wherein the method corrects mutations or defects in USH2A.

272. The method of any one of claims 250-271, wherein the method causes trans-splicing of one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

273. The method of claim 272, wherein the method causes reverse splicing of one or more of USH2A, GPR98 and DFNB31.

274. The method of claim 273, wherein the method causes a reverse splicing of the USH2A.

275. The method of any one of claims 250-274, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa, hearing loss or impairment, night blindness and peripheral vision loss or impairment.

276. The method of any one of claims 250-275, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa and peripheral vision loss or decline.

277. The method of any one of claims 250-276, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa.

278. The method of any one of claims 250-277, wherein the method treats, improves or prevents hearing loss or impairment.

279. The method of any one of claims 250-278, wherein the method treats, improves or prevents vision loss or impairment.

280. The method of any one of claims 250-279, wherein the method treats, improves or prevents one or more of night blindness and peripheral vision loss or decline.

281. The composition or system of any one of claims 1-280, wherein the composition or system further comprises a repair RNA (repRNA) sequence.

282. The composition of claim 281, wherein the reverse splicing system comprises a splice donor, a splice acceptor, and replaces internal exons.

283. The composition of claim 282, wherein the repRNA is operatively ligated to the RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule or the gRNA.

284. A system for targeting nucleic acids to perform trans-splicing, the system comprising: (a) The endonuclease and optionally the RNA molecule as described in any one of claims 1-241, wherein the RNA molecule comprises a sequence complementary to one strand of the target nucleic acid molecule; (b) An RNA-binding polypeptide associated with the said endonuclease; and (c) A repair RNA (repRNA) sequence comprising: (i) One or more exons and / or introns; (ii) Splice donor and / or splice acceptor.

285. The system of claim 284, wherein the RNA molecule is gRNA.

286. The system of claim 284 or 285, wherein the endonuclease is not linked, associated with, and / or fused with an RNA-binding protein.

287. A system for targeting nucleic acids for trans-splicing, the system comprising: (a) The endonuclease and RNA molecule as described in any one of claims 1-241, wherein the RNA molecule comprises a sequence complementary to one strand of the target nucleic acid molecule; and (b) A repair RNA (repRNA) sequence comprising: (i) One or more exons and / or introns; (ii) Splice donor and / or splice acceptor.

288. The system of claim 287, wherein the RNA molecule is gRNA.

289. The system of claim 287 or 288, wherein the endonuclease is not linked, associated with, and / or fused with an RNA-binding protein.

290. The composition of any one of claims 1-241, wherein the composition comprises gRNA, repRNA, and Cas endonuclease operably linked to a single promoter or a bidirectional promoter.

291. The composition of claim 290, wherein the gRNA and repRNA are located on the first side of the bidirectional promoter, and the Cas endonuclease is located on the second side of the bidirectional promoter.

292. A kit comprising a container, the kit comprising a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, lipid nanoparticles as described in claims 233-234, or cells as described in any one of claims 242-247, and instructions for trans-splicing the nucleic acid.

293. A method for targeted trans-splicing of USH2A precursor mRNA in cells, the method comprising contacting the cells with a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, a lipid nanoparticle as described in claims 233-234, or a cell as described in any one of claims 242-247.

294. A method of treating a patient suffering from a disease associated with a mutation in the USH2A gene, the method comprising administering a therapeutically effective amount of a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, lipid nanoparticles as described in claims 233-234, or cells as described in any one of claims 242-247.

295. A method for treating, improving, or preventing a disease associated with a USH2A gene mutation, the method comprising: (a) Contacting cells with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticles of any one of claims 233-234, or the cells of any one of claims 242-247, and (b) Administer an effective amount of the cells to the subject.

296. A method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising administering to the subject an effective amount of a composition or system as described in any one of claims 1-229, a nucleic acid as described in any one of claims 235-241, a viral vector as described in any one of claims 231-232, a lipid nanoparticle as described in claims 233-234, or a cell as described in any one of claims 242-247.

297. A method for treating, improving, or preventing Usher syndrome or its symptoms in a subject of need, the method comprising: (a) Contacting cells with the composition or system of any one of claims 1-229, the nucleic acid of any one of claims 235-241, the viral vector of any one of claims 231-232, the lipid nanoparticles of any one of claims 233-234, or the cells of any one of claims 242-247, and (b) Administer an effective amount of the cells to the subject.

298. The method of any one of claims 295 or 297, wherein the cells are derived from the subject.

299. The method of any one of claims 295-298, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II or Usher syndrome type III.

300. The method of claim 299, wherein the Usher syndrome is Usher syndrome type I.

301. The method of claim 299, wherein the Usher syndrome is Usher syndrome type II.

302. The method of claim 299, wherein the Usher syndrome is Usher syndrome type III.

303. The method of any one of claims 295-302, wherein the method targets one or more Usher syndrome-related genes.

304. The method of claim 303, wherein the method targets one or more genes selected from the group consisting of: CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

305. The method of claim 303 or 304, wherein the method targets one or more of USH2A, GPR98 and DFNB31.

306. The method of claim 305, wherein the method targets USH2A.

307. The method of any one of claims 295-306, wherein the method corrects mutations or defects in one or more Usher syndrome-related genes.

308. The method of any one of claims 295-307, wherein the method corrects mutations or defects in one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

309. The method of claim 308, wherein the method corrects mutations or defects in one or more of USH2A, GPR98, and DFNB31.

310. The method of claim 309, wherein the method corrects mutations or defects in USH2A.

311. The method of any one of claims 295-310, wherein the method causes trans-splicing of one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

312. The method of claim 311, wherein the method causes reverse splicing of one or more of USH2A, GPR98 and DFNB31.

313. The method of claim 312, wherein the method causes a reverse splicing of the USH2A.

314. The method of any one of claims 295-313, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa, hearing loss or deafness, night blindness and peripheral vision loss or deafness.

315. The method of any one of claims 295-314, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa and peripheral vision loss or decline.

316. The method of any one of claims 295-315, wherein the method treats, improves or prevents one or more symptoms of retinitis pigmentosa.

317. The method of any one of claims 295-316, wherein the method treats, improves or prevents hearing loss or impairment.

318. The method of any one of claims 295-317, wherein the method treats, improves or prevents vision loss or impairment.

319. The method of any one of claims 295-318, wherein the method treats, improves or prevents one or more of night blindness and peripheral vision loss or decline.

320. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif has the binding SEQ ID NO: 2023 or SEQ ID NO: The molecular activity of the USH2A target nucleic acid in 2024, said molecular activity being greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, said multiple being measured based on the trans-splicing editing rate relative to the non-target rate.

321. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif is selected from any one of SEQ ID NO: 804-2022.

322. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticles of any one of claims 233-234; or the cells of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the USH2A target nucleic acid of SEQ ID NO: 2023 or SEQ ID NO: 2024 is associated with SEQ ID NO: 2024. The molecular activity of any of the compounds in NO:804-2022 is greater than a multiple of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or about 7.5, wherein the multiple change is measured based on the trans-splicing editing rate relative to the non-targeting rate.

323. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif is bound to SEQ ID NO: The intron positions of the USH2A target nucleic acid in 2024, wherein the intron positions are approximately 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 5 00, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000, optionally wherein the binding motif is selected from SEQ Any of the SEQ ID NO: 804-2022 and bind to position 13 of intron 13 of the USH2A target nucleic acid (SEQ ID NO: 2024).The location relative to the splice donor site is selected from, but not limited to, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 mm from the splice donor site in intron 13 of USH2A. 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or approximately 1000 nucleotides.

324. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif binds to intron 13 of USH2A.

325. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif binds to an exon position of the USH2A target, the exon position being approximately at position 0, -10, -20, -30, -40, -50, -60, -70, or approximately -80.

326. The composition or system of any one of claims 1-230, 281-283, or 290-291; the nucleic acid of any one of claims 235-241; the viral vector of any one of claims 231-232; the lipid nanoparticle of any one of claims 233-234; or the cell of any one of claims 242-247; the pharmaceutical composition of any one of claims 248; the kit of any one of claims 249 or 292; the method of any one of claims 250-280 or 293-319; or the system of any one of claims 284-289, wherein the binding motif binds to position 13 of exon 13 of the USH2A target nucleic acid (SEQ ID NO: 2023), wherein the position is selected from about position -10, -20, -30, -40, -50, -60, -70, or about -80, optionally wherein the binding motif is selected from any one of SEQ ID NO: 804-2022 and binds to the USH2A target nucleic acid (SEQ ID NO: 2023). The position of exon 13 of NO:2023, wherein the position relative to the splice donor site is selected from, but not limited to, approximately position -10, -20, -30, -40, -50, -60, -70, or approximately -80.

327. A repRNA polynucleotide comprising a binding motif for a target nucleic acid, said target nucleic acid being intron 13 of human USH2A or exon 13 of human USH2A.

328. The repRNA of claim 327, wherein exon 13 of human USH2A is or comprises a polynucleotide sequence of SEQ ID NO: 2023, and / or intron 13 of human USH2A is or comprises a polynucleotide sequence of SEQ ID NO: 2024.

329. The repRNA of claim 327 or 328, wherein the binding motif is selected from a polynucleotide having a nucleic acid sequence selected from any of SEQ ID NO:804-2022, or a fragment or variant thereof, optionally having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with it and / or having about 1 to about 20 (e.g., about 1, or about 2, or about 3, or about 4, or about 5) nucleic acid modifications, said nucleic acid modifications optionally selected from substitution, addition, or deletion.

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